Figure 2 ( A ) Representative images showing the localization of AID ( green ) , AIDR190X ( green ) , and endogenous IgH loci ( red ) in nucleofected Aicda - / - CH12F3 cells . The IgH loci are visualized using Sμ - gRNA and indicated by white arrows . An illustration of CRISPR - Sirius IgH locus visualization is shown on top . Scale bar , 5 µm . ( B ) OptoDroplet formation in CH12F3 cells . Schematic illustration of light - induced optoDroplet system is depicted on top . AID variants were tagged to the N terminus of the optoDroplet construct and blue light was employed to promote the formation of puncta three times followed by imaging each time . Representative images after the third blue light activation in Aicda - / - CH12F3 cells with the indicated AID variants are shown at the bottom . White dashed line and green line depict the shapes of nucleus and nucleolus based on Hoechst staining or GFP - FBL , respectively . Scale bar , 5 µm . ( C ) Representative images showing optoDroplet formation of indicated AID variants in HEK293T cells . CRM1 inhibitor ( CRM1i ) was used for selective trapping of AID in nuclei . Scale bar , 5 µm . ( D ) The fluorescence intensity of indicated AID variants in each HEK293T cell nucleus was measured and normalized relative to the fluorescence intensity of the whole cell . Data are shown as box plots . Values between lower quartile and upper quartile are represented by box ranges , a horizontal line within the box represents the median , and whisker extends from the minimum value to the maximum value . " + " indicates the mean level . Each dot indicates one cell , and more than 20 cells represent two biological replicate samples were counted . One - way ANOVA followed by Dunnett ' s multiple comparisons test was performed . * * * * , p < 0 . 0001 ; * , p < 0 . 05 ; ns , p > 0 . 05 . ( E ) Intensity recovery lines for optoAID ∆ C droplets in FRAP experiment in HEK293T cells . Bleaching occurs at t = 0 s . The background - subtracted fluorescence intensities are normalized by pre - bleach values . The mean with SD of 10 pairs of optoAIDR190X and 10 pairs of optoAIDcry is shown , respectively . ( F ) Formation of puncta in primary B cells complemented with AID - FUS - GFP or AID - FUSm - GFP . FUSm contains 13 mutations on either Y or R residue . Left : Representative images showing cellular localization of AID - FUS - GFP and AID - FUSm - GFP with and without CRM1 inhibitor ( CRM1i ) treatment in Aicda - / - primary B cells . Scale bar , 5 µm . Right : Quantification of the number of cells with condensed puncta ( 3 biological replicates ) . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 . ( G ) CSR levels to IgG1 in activated Aicda - / - primary B cells complemented with the indicated protein . Left , representative flow cytometry plots measuring CSR to IgG1 . Co - expressed GFP was used to indicate the infected fractions . Right , the mean with SD of three biological replicates is shown in the bar plot . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative images showing the localization of AID (green), AIDR190X (green), and endogenous IgH loci (red) in nucleofected Aicda-/- CH12F3 cells. The IgH loci are visualized using Sμ-gRNA and indicated by white arrows. An illustration of CRISPR-Sirius IgH locus visualization is shown on top. Scale bar, 5 µm.(B) OptoDroplet formation in CH12F3 cells. Schematic illustration of light-induced optoDroplet system is depicted on top. AID variants were tagged to the N terminus of the optoDroplet construct and blue light was employed to promote the formation of puncta three times followed by imaging each time. Representative images after the third blue light activation in Aicda-/- CH12F3 cells with the indicated AID variants are shown at the bottom. White dashed line and green line depict the shapes of nucleus and nucleolus based on Hoechst staining or GFP-FBL, respectively. Scale bar, 5 µm.(C) Representative images showing optoDroplet formation of indicated AID variants in HEK293T cells. CRM1 inhibitor (CRM1i) was used for selective trapping of AID in nuclei. Scale bar, 5 µm.(D) The fluorescence intensity of indicated AID variants in each HEK293T cell nucleus was measured and normalized relative to the fluorescence intensity of the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell, and more than 20 cells represent two biological replicate samples were counted. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ****, p<0.0001; *, p<0.05; ns, p>0.05.(E) Intensity recovery lines for optoAID∆C droplets in FRAP experiment in HEK293T cells. Bleaching occurs at t = 0 s. The background-subtracted fluorescence intensities are normalized by pre-bleach values. The mean with SD of 10 pairs of optoAIDR190X and 10 pairs of optoAIDcry is shown, respectively.(F) Formation of puncta in primary B cells complemented with AID-FUS-GFP or AID-FUSm-GFP. FUSm contains 13 mutations on either Y or R residue. Left: Representative images showing cellular localization of AID-FUS-GFP and AID-FUSm-GFP with and without CRM1 inhibitor (CRM1i) treatment in Aicda-/- primary B cells. Scale bar, 5 µm. Right: Quantification of the number of cells with condensed puncta (3 biological replicates). Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.(G) CSR levels to IgG1 in activated Aicda-/- primary B cells complemented with the indicated protein. Left, representative flow cytometry plots measuring CSR to IgG1. Co-expressed GFP was used to indicate the infected fractions. Right, the mean with SD of three biological replicates is shown in the bar plot. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


Figure 3 ( A ) Representative immunofluorescence images of endogenous AID in the presence of ectopically - expressed EV , AID , AIDR190X , or AIDcry in cytokine - stimulated CH12F3 cells . EV , empty vector control . An antibody against AID C terminus ( 185 - 198 aa ) detected both endogenous and ectopically - expressed full - length AID . The nucleolus is indicated by co - nucleofected mCherry - FBL . The white dashed line depicts the shape of the nucleus based on Hoechst staining . Arrows indicate the location of puncta formed by endogenous AID . The images were taken by the N - SIM microscope and processed via N - SIM software . A middle slice of the images was shown . Scale bar , 5 µm . ( B ) Co - condensation of AID and AIDR190X in the nucleus of CH12F3 cells in the early G1 phase . Top : experimental layout . GFP - tagged wild - type AID ( green ) and mCherry - tagged AID variants ( red ) were co - nucleofected in Aicda - / - CH12F3 cells . The resulting cells were arrested with nocodazole for 12 hours after transfection . Six hours later , nocodazole was removed from the system for 1 . 5 hours before imaging . Bottom left : representative images showing subcellular localization of indicated AID variants . The white dashed line depicts the shape of the nucleus based on Hoechst staining . The white arrow indicates the location of the co - condensed puncta . Scale bar , 5 µm . Bottom right : plot profiles of the images . Cyto : cytoplasm , Nuc : nucleus . ( C ) Quantification of the relative fluorescence intensity of AID - GFP in the diluted phase of cells shown in ( B ) . The fluorescence intensity of AID - GFP in the diluted nuclear phase was normalized by the total intensity from the whole cell . Data are shown as box plots . Values between lower quartile and upper quartile are represented by box ranges , a horizontal line within the box represents the median , and whisker extends from the minimum value to the maximum value . " + " indicates the mean level . Each dot indicates one cell ( n = 25 ) , and representative result from three biological replicates were shown . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 . ( D ) Dynamics of trapping of wild - type AID in optoAIDR190X condensates . Top , representative fluorescence images at different time points . Dashed circles indicate the co - condensed puncta formation of AID - GFP . Scale bar , 5 µm . Bottom , a summary of 12 observed cells is shown as mean ± SD in a bar graph . Percentages of optoAIDR190X puncta with trapped wild - type AID are plotted for the second and third blue - light activations , and the colored dot indicates one cell .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Representative immunofluorescence images of endogenous AID in the presence of ectopically-expressed EV, AID, AIDR190X, or AIDcry in cytokine-stimulated CH12F3 cells. EV, empty vector control. An antibody against AID C terminus (185-198 aa) detected both endogenous and ectopically-expressed full-length AID. The nucleolus is indicated by co-nucleofected mCherry-FBL. The white dashed line depicts the shape of the nucleus based on Hoechst staining. Arrows indicate the location of puncta formed by endogenous AID. The images were taken by the N-SIM microscope and processed via N-SIM software. A middle slice of the images was shown. Scale bar, 5 µm.(B) Co-condensation of AID and AIDR190X in the nucleus of CH12F3 cells in the early G1 phase. Top: experimental layout. GFP-tagged wild-type AID (green) and mCherry-tagged AID variants (red) were co-nucleofected in Aicda-/- CH12F3 cells. The resulting cells were arrested with nocodazole for 12 hours after transfection. Six hours later, nocodazole was removed from the system for 1.5 hours before imaging. Bottom left: representative images showing subcellular localization of indicated AID variants. The white dashed line depicts the shape of the nucleus based on Hoechst staining. The white arrow indicates the location of the co-condensed puncta. Scale bar, 5 µm. Bottom right: plot profiles of the images. Cyto: cytoplasm, Nuc: nucleus.(C) Quantification of the relative fluorescence intensity of AID-GFP in the diluted phase of cells shown in (B). The fluorescence intensity of AID-GFP in the diluted nuclear phase was normalized by the total intensity from the whole cell. Data are shown as box plots. Values between lower quartile and upper quartile are represented by box ranges, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. "+" indicates the mean level. Each dot indicates one cell (n = 25), and representative result from three biological replicates were shown. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.(D) Dynamics of trapping of wild-type AID in optoAIDR190X condensates. Top, representative fluorescence images at different time points. Dashed circles indicate the co-condensed puncta formation of AID-GFP. Scale bar, 5 µm. Bottom, a summary of 12 observed cells is shown as mean ±SD in a bar graph. Percentages of optoAIDR190X puncta with trapped wild-type AID are plotted for the second and third blue-light activations, and the colored dot indicates one cell.


Figure 4 ( B ) The optoDroplet formation of indicated AID mutants along with light stimulation . Top , the procedure of optoDroplet assay is illustrated with indicated light stimulation ( blue arrow ) and image acquisition ( black arrow ) time points . Bottom , line plots show quantification of cells with optoDroplet formation as mean ± SD at each acquisition time point . The total acquired cell numbers of each genotype are indicated in parentheses . Significance assessment between AIDR190X and AIDR190X - 4R was shown at each time point . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 . ( C ) CSR levels of ectopically - expressed AID variants in AID - proficient CH12F3 cells . Left , representative flow cytometry plots of CSR to IgA . Co - expressed GFP indicates the infected cell fractions . Right , the CSR mean with SD of three biological replicates is shown in the bar plot . ( D ) CSR levels of ectopically - expressed AID variants in CSR - activated B cells . Left , representative flow cytometry plots of CSR to IgG1 . Right , the bar plot shows the CSR mean with SD of four biological replicates . Data information : One - way ANOVA followed by Dunnett ' s multiple comparisons test was performed for ( C ) and ( D ) . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) The optoDroplet formation of indicated AID mutants along with light stimulation. Top, the procedure of optoDroplet assay is illustrated with indicated light stimulation (blue arrow) and image acquisition (black arrow) time points. Bottom, line plots show quantification of cells with optoDroplet formation as mean ± SD at each acquisition time point. The total acquired cell numbers of each genotype are indicated in parentheses. Significance assessment between AIDR190X and AIDR190X-4R was shown at each time point. Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.(C) CSR levels of ectopically-expressed AID variants in AID-proficient CH12F3 cells. Left, representative flow cytometry plots of CSR to IgA. Co-expressed GFP indicates the infected cell fractions. Right, the CSR mean with SD of three biological replicates is shown in the bar plot. (D) CSR levels of ectopically-expressed AID variants in CSR-activated B cells. Left, representative flow cytometry plots of CSR to IgG1. Right, the bar plot shows the CSR mean with SD of four biological replicates. Data information: One-way ANOVA followed by Dunnett's multiple comparisons test was performed for (C) and (D). ***, p<0.001; **, p<0.01; *, p<0.05.


Figure 5 ( A ) Left , representative images of MBP - GFP - AID in the presence of 10 % PEG8000 . Scale bar , 10 µm and 2 µm , respectively . Right , the schematic protein sequences . ( B ) Left , representative images of MBP - GFP - AIDcry + CTT or MBP - GFP - AIDcry in the presence 10 % PEG8000 at the indicated protein concentrations . Scale bar , 10 µm . Right , sizes of MBP - GFP - AIDcry + CTT or MBP - GFP - AIDcry protein condensates are plotted as box plots with three technical replicates . The box represents values between the lower quartile and the upper quartile , a horizontal line within the box represents the median , and whisker extends from the minimum value to the maximum value . All points are listed and mean is indicated by " + " . Unpaired two - tailed Student ' s t - test was performed . * * * * , p < 0 . 0001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Left, representative images of MBP-GFP-AID in the presence of 10% PEG8000. Scale bar, 10 µm and 2 µm, respectively. Right, the schematic protein sequences.(B) Left, representative images of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry in the presence 10% PEG8000 at the indicated protein concentrations. Scale bar, 10 µm. Right, sizes of MBP-GFP-AIDcry+CTT or MBP-GFP-AIDcry protein condensates are plotted as box plots with three technical replicates. The box represents values between the lower quartile and the upper quartile, a horizontal line within the box represents the median, and whisker extends from the minimum value to the maximum value. All points are listed and mean is indicated by "+". Unpaired two-tailed Student's t-test was performed. ****, p<0.0001.


Figure 6 ( A ) AID deamination assay was performed in vitro . Left , a schematic diagram of deamination activity assay . Right , the deamination reaction with indicated protein combinations was processed and visualized . " + " : 0 . 25 μM of indicated protein ; " + + " : 0 . 5 μM of indicated protein ; " - " : not added . ( B ) CSR to IgA induced by AID variants in CH12F3 cells . Left , representative flow cytometry plots . Right , the CSR mean with SD of three biological replicates is shown in the bar plot . One - way ANOVA followed by Dunnett ' s multiple comparisons test was performed . * * * , p < 0 . 001 ; * * , p < 0 . 01 . ( C ) Representative images of the co - condensation of MBP - mCherry - AIDcry and MBP - GFP - AID . The protein concentrations and molecular ratios are indicated . Scale bar , 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) AID deamination assay was performed in vitro. Left, a schematic diagram of deamination activity assay. Right, the deamination reaction with indicated protein combinations was processed and visualized. "+": 0.25 μM of indicated protein; "++": 0.5 μM of indicated protein; "-": not added.(B) CSR to IgA induced by AID variants in CH12F3 cells. Left, representative flow cytometry plots. Right, the CSR mean with SD of three biological replicates is shown in the bar plot. One-way ANOVA followed by Dunnett's multiple comparisons test was performed. ***, p<0.001; **, p<0.01.(C) Representative images of the co-condensation of MBP-mCherry-AIDcry and MBP-GFP-AID. The protein concentrations and molecular ratios are indicated. Scale bar, 10 µm.


Figure 1 ( C ) Immunocytochemistry showing post - synaptic marker PSD - 95 ( red ) , pre - synaptic marker Synapsin - I ( green ) , dendritic marker Map2 ( blue ) and co - localization of PSD - 95 and Synapsin - I from dissociated Nedd4 - 2 WT or Nedd4 - 2 cKO hippocampal neurons at DIV 16 . Representative secondary dendrites ( left ) and quantification of colocalized synaptic puncta number and area ( right ) ( n = 36 for Nedd4 - 2 WT and Nedd4 - 2 cKO neurons ) are shown . Scale bar : 5 µm . ( F ) A train of high frequency stimulation ( HFS ; 100 Hz for 1s ) induced early - phase LTP ( E - LTP ) at Schaffer collateral synapses in Nedd4 - 2 WT ( n = 7 slices from 6 mice ) and Nedd4 - 2 cKO ( n = 6 slices from 5 mice ) mice . Representative fEPSP traces were recorded before and 60 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing the fEPSP slopes measured 50 - 60 min after a HFS at Schaffer collateral synapses was on the right . ( G ) Late - phase LTP ( L - LTP ) induced by four trains of HFS in Nedd4 - 2 WT slices ( n = 7 slices from 7 mice ) and Nedd4 - 2 cKO slices ( n = 9 slices from 8 mice ) . Representative fEPSP traces were recorded before and 180 min after LTP induction ( Scale bars : 0 . 5 mV , 5 ms ) . Summary bar graphs showing the fEPSP slopes measured 160 - 180 min after L - LTP induction at Schaffer collateral synapses was on the right .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from dissociated Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons at DIV 16. Representative secondary dendrites (left) and quantification of colocalized synaptic puncta number and area (right) (n = 36 for Nedd4-2 WT and Nedd4-2 cKO neurons) are shown. Scale bar: 5 µm.(F) A train of high frequency stimulation (HFS; 100 Hz for 1s) induced early-phase LTP (E-LTP) at Schaffer collateral synapses in Nedd4-2 WT (n = 7 slices from 6 mice) and Nedd4-2 cKO (n = 6 slices from 5 mice) mice. Representative fEPSP traces were recorded before and 60 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 50-60 min after a HFS at Schaffer collateral synapses was on the right. (G) Late-phase LTP (L-LTP) induced by four trains of HFS in Nedd4-2 WT slices (n = 7 slices from 7 mice) and Nedd4-2 cKO slices (n = 9 slices from 8 mice). Representative fEPSP traces were recorded before and 180 min after LTP induction (Scale bars: 0.5 mV, 5 ms). Summary bar graphs showing the fEPSP slopes measured 160-180 min after L-LTP induction at Schaffer collateral synapses was on the right.


Figure 2 ( B ) Barnes maze test from Nedd4 - 2 WT ( n = 8 ) or Nedd4 - 2 cKO ( n = 8 ) mice ( 6 - 8 weeks old ) . Schematic representation of Barnes maze ( top left ) . Mice were trained for 4 days with three training trials on each day to locate the escape box ( black circle ) . Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping ( top right ) . Schematic representation of Barnes maze on probe trials ( bottom left ) . Quadrant occupancy on probe trial at day5 and at day12 ( bottom right ) were assessed by the time spent in the target area ( green quadrant ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(B) Barnes maze test from Nedd4-2 WT (n = 8) or Nedd4-2 cKO (n = 8) mice (6-8 weeks old). Schematic representation of Barnes maze (top left). Mice were trained for 4 days with three training trials on each day to locate the escape box (black circle). Learning curve during 4 training days was shown by the escape latency and the number of errors before escaping (top right). Schematic representation of Barnes maze on probe trials (bottom left). Quadrant occupancy on probe trial at day5 and at day12 (bottom right) were assessed by the time spent in the target area (green quadrant).


Figure 3 ( B ) Western blots of F - actin to G - actin ratio with or without cLTP induction from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured neurons . Schematic representation of the experimental design for cLTP inductions ( top left ) . Representative western blots ( bottom left ) and quantification of F - actin to G - actin from Nedd4 - 2 WT or Nedd4 - 2 cKO ( right ) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis ( n = 11 and 8 for Nedd4 - 2 WT and Nedd4 - 2 cKO cultures , respectively ) . For quantification , data from cLTP induction were normalized to data from control ( CTL ) condition .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Western blots of F-actin to G-actin ratio with or without cLTP induction from Nedd4-2 WT or Nedd4-2 cKO cultured neurons. Schematic representation of the experimental design for cLTP inductions (top left). Representative western blots (bottom left) and quantification of F-actin to G-actin from Nedd4-2 WT or Nedd4-2 cKO (right) cultures after cLTP induction and recovery in normal aCSF for 10 or 30 min before lysis (n = 11 and 8 for Nedd4-2 WT and Nedd4-2 cKO cultures, respectively). For quantification, data from cLTP induction were normalized to data from control (CTL) condition.


Figure 4 ( A ) Western blots of Nedd4 - 2 , total cofilin , phospho ( p ) - cofilin and GAPDH from Nedd4 - 2 WT or Nedd4 - 2 cKO forebrains at postnatal day 14 . Representative western blots and quantification of total cofilin relative to GAPDH and p - cofilin relative to total cofilin ( n = 6 for Nedd4 - 2 WT and Nedd4 - 2 cKO brains ) were shown . ( B ) Immunocytochemistry of cofilin , p - cofilin and dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons at DIV 16 . Representative dendrites of Nedd4 - 2 WT and Nedd4 - 2 cKO neurons and quantification of total cofilin relative to MAP2 and p - cofilin relative to total cofilin are shown ( n = 25 and 26 for Nedd4 - 2 WT and Nedd4 - 2 cKO neurons , respectively ) . Scale bar : 5 µm . ( C ) Immunocytochemistry showing cofilin , p - cofilin , and dendritic marker MAP2 from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16 . Representative dendrites and quantification from Nedd4 - 2 WT or Nedd4 - 2 cKO cultures are shown ( n = 36 - 39 neurons ) . Scale bar : 5 µm . For quantification , data from cLTP induction were normalized to data from control ( CTL ) condition .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Western blots of Nedd4-2, total cofilin, phospho (p)-cofilin and GAPDH from Nedd4-2 WT or Nedd4-2 cKO forebrains at postnatal day 14. Representative western blots and quantification of total cofilin relative to GAPDH and p-cofilin relative to total cofilin (n = 6 for Nedd4-2 WT and Nedd4-2 cKO brains) were shown.(B) Immunocytochemistry of cofilin, p-cofilin and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16. Representative dendrites of Nedd4-2 WT and Nedd4-2 cKO neurons and quantification of total cofilin relative to MAP2 and p-cofilin relative to total cofilin are shown (n = 25 and 26 for Nedd4-2 WT and Nedd4-2 cKO neurons, respectively). Scale bar: 5 µm.(C) Immunocytochemistry showing cofilin, p-cofilin, and dendritic marker MAP2 from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons induced with or without cLTP at DIV 16. Representative dendrites and quantification from Nedd4-2 WT or Nedd4-2 cKO cultures are shown (n = 36-39 neurons). Scale bar: 5 µm. For quantification, data from cLTP induction were normalized to data from control (CTL) condition.


Figure 5 ( A ) Immunocytochemistry showing post - synaptic marker PSD - 95 ( red ) , pre - synaptic marker Synapsin - I ( green ) , dendritic marker Map2 ( blue ) and co - localization of PSD - 95 and Synapsin - I from Nedd4 - 2 WT or Nedd4 - 2 cKO cultured hippocampal neurons at DIV 16 treated with TAT , TAT - S3 , or TAT - pS3 peptide for 12 hrs ( 10 μM ) . Representative secondary dendrites of Nedd4 - 2 WT ( top left ) and Nedd4 - 2 cKO ( bottom left ) neurons are shown . Quantification of colocalized synaptic puncta number and synaptic puncta area for Nedd4 - 2 WT ( top right ) and Nedd4 - 2 cKO ( bottom right ) neurons is on the right ( n = 32 - 36 neurons ) . Scale bar : 5 µm . ( B ) Patch - clamp recordings from Nedd4 - 2 WT or Nedd4 - 2 cKO hippocampal neurons treated with TAT , TAT - S3 , or TAT - pS3 peptide for 12 hrs ( 10 μM ) at DIV 14 - 17 . Representative mEPSC traces from Nedd4 - 2 WT ( top left ) and Nedd4 - 2 cKO ( bottom left ) are shown . Quantification of mEPSC amplitude and frequency from Nedd4 - 2 WT ( top right ) and Nedd4 - 2 cKO ( bottom right ) neurons are shown ( n = 22 - 24 neurons ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Immunocytochemistry showing post-synaptic marker PSD-95 (red), pre-synaptic marker Synapsin-I (green), dendritic marker Map2 (blue) and co-localization of PSD-95 and Synapsin-I from Nedd4-2 WT or Nedd4-2 cKO cultured hippocampal neurons at DIV 16 treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM). Representative secondary dendrites of Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) neurons are shown. Quantification of colocalized synaptic puncta number and synaptic puncta area for Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons is on the right (n = 32-36 neurons). Scale bar: 5 µm.(B) Patch-clamp recordings from Nedd4-2 WT or Nedd4-2 cKO hippocampal neurons treated with TAT, TAT-S3, or TAT-pS3 peptide for 12 hrs (10 μM) at DIV 14-17. Representative mEPSC traces from Nedd4-2 WT (top left) and Nedd4-2 cKO (bottom left) are shown. Quantification of mEPSC amplitude and frequency from Nedd4-2 WT (top right) and Nedd4-2 cKO (bottom right) neurons are shown (n = 22-24 neurons).


Figure 6 ( C ) Barnes maze test after intraperitoneally injecting TAT ( n = 8 ) or TAT - pS3 ( n = 8 ) peptide in Nedd4 - 2 WT mice ( top ) , or TAT ( n = 9 ) or TAT - pS3 ( n = 8 ) peptides in Nedd4 - 2 cKO mice ( bottom ) . Learning curve during 4 training days of Barnes maze task showing the escape latency , number of errors before escaping , and the quadrant occupancy on probe trial at day 5 and at day 12 were assessed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(C) Barnes maze test after intraperitoneally injecting TAT (n = 8) or TAT-pS3 (n = 8) peptide in Nedd4-2 WT mice (top), or TAT (n = 9) or TAT-pS3 (n = 8) peptides in Nedd4-2 cKO mice (bottom). Learning curve during 4 training days of Barnes maze task showing the escape latency, number of errors before escaping, and the quadrant occupancy on probe trial at day 5 and at day 12 were assessed.


Figure 1 ( B ) Immunoblot analysis of membrane fractions of HEK293 Flp - In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane - bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor . An isotype antibody was used as a negative control . Calnexin served as a loading control . Levels of membrane - bound TREM2 were quantified by MSD ELISA . Data represent the mean ± SEM ( n = 3 ) . One - way ANOVA , Tukey ' s post hoc test ; p ( DMSO vs GM ) = 0 . 0011 ; p ( DMSO vs isotype ) = 0 . 992 ; p ( isotype vs 4D9 ) = 0 . 0005 ; n . s . , not significant . ( C ) Immunoblot analysis of conditioned media from HEK293 Flp - In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor . An isotype antibody was used as a negative control . sAPPα served as a loading control . Note that heavy and light chains of the antibodies used for treatment are also detected and annotated . Levels of sTREM2 were quantified by MSD ELISA . Data represent the mean ± SEM ( n = 3 ) . One - way ANOVA , Tukey ' s post hoc test ; p ( DMSO vs GM ) < 0 . 0001 ; p ( DMSO vs isotype ) = 0 . 6372 ; p ( isotype vs 4D9 ) < 0 . 0001 ; n . s . , not significant . ( D ) 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp - In cells stably overexpressing mouse TREM2 and mouse DAP12 . An anti - HA antibody was used as a positive control while empty vector transfected HEK293 Flp - In cells were used as a negative control . Scale bar = 10µm . ( F ) Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope ( upper panel ) . Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 ( lower panel ) . ( G ) 4D9 binding to mouse TREM2 ECD is competed off by a stalk region peptide . A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1 . 3uM . Data represent the mean ± SEM ( n = 3 ) . ( H ) Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ectodomain evaluated by BIAcore , Kon = 5 . 9 x 105 M - 1s - 1 , Koff = 4 . 0 x 10 - 5 s - 1 , KD = 68 pM . 4D9 binding to human TREM2 or mouse TREM1 was undetectable .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Immunoblot analysis of membrane fractions of HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) = 0.0011; p (DMSO vs isotype) = 0.992; p (isotype vs 4D9) = 0.0005; n.s., not significant.(C) Immunoblot analysis of conditioned media from HEK293 Flp-In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM (n=3). One-way ANOVA, Tukey's post hoc test; p (DMSO vs GM) < 0.0001; p (DMSO vs isotype) = 0.6372; p (isotype vs 4D9) < 0.0001; n.s., not significant.(D) 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12. An anti-HA antibody was used as a positive control while empty vector transfected HEK293 Flp-In cells were used as a negative control. Scale bar= 10µm.(F) Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope (upper panel). Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 (lower panel).(G) 4D9 binding to mouse TREM2 ECD is competed off by a stalk region peptide. A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1.3uM. Data represent the mean ± SEM (n=3).(H) Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ectodomain evaluated by BIAcore, Kon = 5.9 x 105 M-1s-1, Koff = 4.0 x 10-5 s-1, KD = 68 pM. 4D9 binding to human TREM2 or mouse TREM1 was undetectable.


Figure 2 ( A ) Flow cytometry dose response curve for cell binding of 4D9 mAb ( EC50 = 0 . 29nM ) , 4D9 Fab ( EC50 = 0 . 17nM ) , and isotype to HEK cells stably overexpressing mouse TREM2 . Data represent the mean ± SEM ( n = 2 ) . ( B ) In vitro ADAM17 sheddase activity is blocked by 4D9 - effectorless mAb and 4D9 Fab fragment but not an isotype control . Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb , 4D9 Fab and isotype control . Data represent the mean ± SEM ( n = 6 ) . One - way ANOVA , Tukey ' s post hoc test ; p ( 4D9 Fab vs 4D9 mAb ) = 0 . 8855 ; p ( 4D9 Fab vs uncleaved ) < 0 . 0001 ; p ( 4D9 mAb vs uncleaved ) < 0 . 0001 ; n . s . , not significant . ( C ) ELISA - mediated quantification of sTREM2 in conditioned media from ( D ) AlphaLISA - mediated quantification of pSYK levels in HEK293 Flp - In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb , 4D9 Fab or isotype for 5 minutes . Data represent the mean ± SEM ( n = 3 ) . ( E ) AlphaLISA - mediated quantification of pSYK levels in HEK293 Flp - In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg / ml POPC / POPS liposomes and 20µg / ml 4D9 mAb , 4D9 Fab or isotype for 5 minutes . pSYK levels were also determined for cells treated with liposomes only . Data represent the mean ± SEM ( n = 3 ) . Two - way ANOVA , Tukey ' s post hoc test ( cell line effect : F1 , 16 = 365 . 7 , p = < 0 . 0001 ; treatment effect : F3 , 16 = 39 . 35 , p < 0 . 0001 ; cell line x treatment effect : F3 , 16 = 38 . 75 , p < 0 . 0001 ) ; p ( no Ab vs isotype ) = 0 . 6218 ; p ( no Ab vs 4D9 mAb ) < 0 . 0001 ; p ( no Ab vs 4D9 Fab ) = 0 . 7301 ; p ( isotype vs 4D9 mAb ) < 0 . 0001 ; p ( isotype vs 4D9 Fab ) > 0 . 9999 ; p ( 4D9 mAb vs 4D9 Fab ) < 0 . 0001 ; n . s . , not significant .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Flow cytometry dose response curve for cell binding of 4D9 mAb (EC50= 0.29nM), 4D9 Fab (EC50=0.17nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM (n=2).(B) In vitro ADAM17 sheddase activity is blocked by 4D9-effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab and isotype control. Data represent the mean ± SEM (n=6). One-way ANOVA, Tukey's post hoc test; p (4D9 Fab vs 4D9 mAb) = 0.8855; p (4D9 Fab vs uncleaved) < 0.0001; p (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant.(C) ELISA-mediated quantification of sTREM2 in conditioned media from (D) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab or isotype for 5 minutes. Data represent the mean ± SEM (n=3).(E) AlphaLISA-mediated quantification of pSYK levels in HEK293 Flp-In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1mg/ml POPC/POPS liposomes and 20µg/ml 4D9 mAb, 4D9 Fab or isotype for 5 minutes. pSYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM (n=3). Two-way ANOVA, Tukey's post hoc test (cell line effect: F1,16=365.7, p=<0.0001; treatment effect: F3,16=39.35, p<0.0001; cell line x treatment effect: F3,16=38.75, p<0.0001); p (no Ab vs isotype) = 0.6218; p (no Ab vs 4D9 mAb) < 0.0001; p (no Ab vs 4D9 Fab) = 0.7301; p (isotype vs 4D9 mAb) < 0.0001; p (isotype vs 4D9 Fab) > 0.9999; p (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant.


Figure 3 ( A ) Flow cytometry detection of cell surface binding of 4D9 ( blue ) and isotype ( green ) to wild type and Trem2 - / - mouse bone marrow derived macrophages . Representative histograms are shown as MFI ( mean fluorescent intensity ) . Graphs represent the median signal intensity ± SEM ( n = 3 ) . Student ' s t - test ( two - tailed ) ; Trem2 + / + : p = 0 . 0005 ; Trem2 - / - : p = 0 . 4435 ; n . s . , not significant . ( B ) Immunoblot analysis of membrane fractions of bone marrow derived macrophages ( BMDM ) upon treatment with 4D9 antibody reveals increased levels of membrane - bound TREM2 relative to an isotype antibody . BMDM from TREM2 knockout mice were included to show the specificity of the anti - TREM2 antibody . Calnexin served as a loading control . Levels of membrane - bound TREM2 were quantified by MSD ELISA . TREM2 antibody clone 5F4 was used for detection . Data represent the mean ± SEM ( n = 5 - 6 ) . Unpaired t - test ( two - tailed ) with Welch ' s correction ; p < 0 . 0001 . No gender - specific differences could be observed . ( C ) Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies . BMDM from TREM2 knockout mice were included to show the specificity of the anti - TREM2 antibody . Soluble APP served as a loading control . Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows : Ab hc = antibody heavy chain ; Ab lc = antibody light chain . Levels of sTREM2 were quantified by MSD ELISA . TREM2 antibody clone 5F4 was used for detection . Data represent the mean ± SEM ( n = 6 ) . Unpaired t - test ( two - tailed ) with Welch ' s correction ; p < 0 . 0001 . No gender - specific differences could be observed . ( D ) Wildtype , Trem2 + / - , and Trem2 - / - BMDMs were plated in low concentration of M - CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability . 4D9 and isotype data for each genotype represent the mean ± SEM ( n = 2 and n = 1 , respectively ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Flow cytometry detection of cell surface binding of 4D9 (blue) and isotype (green) to wild type and Trem2-/- mouse bone marrow derived macrophages. Representative histograms are shown as MFI (mean fluorescent intensity). Graphs represent the median signal intensity ± SEM (n=3). Student's t-test (two-tailed); Trem2+/+: p = 0.0005; Trem2-/-: p = 0.4435; n.s., not significant.(B) Immunoblot analysis of membrane fractions of bone marrow derived macrophages (BMDM) upon treatment with 4D9 antibody reveals increased levels of membrane-bound TREM2 relative to an isotype antibody. BMDM from TREM2 knockout mice were included to show the specificity of the anti-TREM2 antibody. Calnexin served as a loading control. Levels of membrane-bound TREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM (n=5-6). Unpaired t-test (two-tailed) with Welch's correction; p<0.0001. No gender-specific differences could be observed.(C) Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies. BMDM from TREM2 knockout mice were included to show the specificity of the anti-TREM2 antibody. Soluble APP served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows: Ab hc= antibody heavy chain; Ab lc= antibody light chain. Levels of sTREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM (n=6). Unpaired t-test (two-tailed) with Welch's correction; p<0.0001. No gender-specific differences could be observed.(D) Wildtype, Trem2+/-, and Trem2-/- BMDMs were plated in low concentration of M-CSF on 4D9 or isotype coated wells for 5 days and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM (n=2 and n=1, respectively).


Figure 4 ( A ) Flow cytometry detection of cell surface 4D9 ( red ) binding and isotype control ( blue ) on primary wild type and Trem2 - / - mouse microglia . Data are shown as MFI ( mean fluorescent intensity ) . ( B ) Uptake assay for fluorescently labelled myelin , Aβ ( 1 - 42 ) and inactivated E . coli particles . The top row shows that myelin and Aβ ( pseudocoloured in white ) accumulate within the plasma membrane of primary microglia ( labeled with dylight - 649 isolectin and pseudocoloured in red ) . Overnight pre - treatment with antibody 4D9 significantly increased the percentage of substrate positive cells ( middle row ) . Pre - treatment with the isotope control did not affect the uptake rate ( bottom row ) . No changes in the uptake of E . coli particles were observed . Substrate uptake in the absence of antibody is shown in the top row . Hoechst 33342 was used to counter stain the nuclei ( in cyan ) and to assess cell density . Scale bar = 20µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Flow cytometry detection of cell surface 4D9 (red) binding and isotype control (blue) on primary wild type and Trem2-/- mouse microglia. Data are shown as MFI (mean fluorescent intensity).(B) Uptake assay for fluorescently labelled myelin, Aβ(1-42) and inactivated E.coli particles. The top row shows that myelin and Aβ (pseudocoloured in white) accumulate within the plasma membrane of primary microglia (labeled with dylight-649 isolectin and pseudocoloured in red). Overnight pre-treatment with antibody 4D9 significantly increased the percentage of substrate positive cells (middle row). Pre-treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E.coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar= 20µm.


Figure 5 ( A ) 4D9 demonstrates standard IgG pharmacokinetics in vivo . Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours , 4 and 7 days after 10 mg / kg intravenous injection of antibody . ( B ) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg / kg and 4D9 - hIgG dosed intravenously at 100 , 50 and 10mg / kg . Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range . Animals were perfused to minimize IgG contribution from the plasma . Data represent the mean ± SEM ( n = 5 ) . ( D ) Target engagement timecourse demonstrated near 100 % 4D9 - bound sTREM2 in CSF of wildtype mice at 24 hrs post dose . Over a 10 day timecourse with timepoints at day 1 , 2 , 4 , 7 and study termination at day 10 , the bound / total sTREM2 reduces gradually to reach ~ 50 % by day 10 . Animals were dosed intravenously with 50mg / kg of isotype and 4D9 antibodies . Data represent the mean ± SEM ( n = 5 ) . ( E ) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg / kg with > 50 % bound sTREM2 at 1mg / kg of antibody . 4D9 - bound sTREM2 was undetectable at 0 . 1mg / kg and lower . 4D9 was IV - dosed at 50 , 10 , 1 , 0 . 1 , 0 . 01 , 0 . 001 , 0 . 0001 mg / kg , and isotype dosed at 50mg / kg . CSF bound : total sTREM2 in wildtype mice was measured at 24hrs . Data represent the mean ± SEM ( n = 5 ) . ( F ) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels . Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD - platform based ELISA . Data represent the mean ± SEM ( n = 5 ) and are shown as pg TREM2 per ug of total protein . One - way ANOVA , Dunnett ' s post hoc test , p ( isotype vs 4D9 ( 100mg / kg ) ) < 0 . 0001 ; p ( isotype vs 4D9 ( 50mg / kg ) ) = 0 . 0007 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) 4D9 demonstrates standard IgG pharmacokinetics in vivo. Peripheral clearance rates of 4D9 on a human IgG effectorless backbone compared to an isotype control were determined in wild type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 hours, 4 and 7 days after 10 mg/kg intravenous injection of antibody.(B) Brain antibody concentration was measured 24hrs post intravenous dosing of isotype hIgG at 100mg/kg and 4D9-hIgG dosed intravenously at 100, 50 and 10mg/kg. Detection of hIgG levels by ELISA demonstrated dose dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM (n=5).(D) Target engagement timecourse demonstrated near 100% 4D9-bound sTREM2 in CSF of wildtype mice at 24 hrs post dose. Over a 10 day timecourse with timepoints at day 1, 2, 4, 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ~50% by day 10. Animals were dosed intravenously with 50mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM (n=5).(E) Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10mg/kg with >50% bound sTREM2 at 1mg/kg of antibody. 4D9-bound sTREM2 was undetectable at 0.1mg/kg and lower. 4D9 was IV-dosed at 50, 10, 1, 0.1, 0.01, 0.001, 0.0001 mg/kg, and isotype dosed at 50mg/kg. CSF bound: total sTREM2 in wildtype mice was measured at 24hrs. Data represent the mean ± SEM (n=5).(F) 4D9 antibody demonstrates dose dependent increase of total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wildtype mice dosed with 4D9 or isotype control was performed by a MSD-platform based ELISA. Data represent the mean ± SEM (n=5) and are shown as pg TREM2 per ug of total protein. One-way ANOVA, Dunnett's post hoc test, p (isotype vs 4D9 (100mg/kg)) < 0.0001; p (isotype vs 4D9 (50mg/kg)) = 0.0007.


Figure 6 ( B ) Immunohistochemical costainings of TREM2 ( magenta ) and IBA1 ( green ) microglia in the cortex of isotype control and 4D9 injected WT and APP - NL - G - F mice . Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes . Scale bar = 10µm ; scale bar ( inset ) = 2 . 5µm . ( C ) Quantification of cortical TREM2 stainings shown in B . Two - way ANOVA , Tukey ' s multiple comparison test ( Genotype effect : F1 , 23 = 70 . 63 , p = < 0 . 0001 ; Treatment effect : F1 , 23 = 14 . 33 , p = 0 . 0010 ; Genotype x Treatment effect : F1 , 23 = 14 . 51 , p = 0 . 0009 ) ; p ( WT isotype vs WT 4D9 ) > 0 . 9999 ; p ( WT isotype vs APP isotype ) = 0 . 02 ; p ( WT 4D9 vs APP 4D9 ) < 0 . 0001 ; p ( APP isotype vs APP 4D9 ) = 0 . 0001 ; n . s . , not significant . ( D ) Quantification of cortical IBA1 staining shown in B . Two - way ANOVA , Tukey ' s multiple comparison test ( Genotype effect : F1 , 21 = 215 . 4 , p = < 0 . 0001 ; Treatment effect : F1 , 21 = 0 . 5994 , p = 0 . 4475 ; Genotype x Treatment effect : F1 , 21 = 0 . 2992 , p = 0 . 5902 ) ; p ( WT isotype vs APP isotype ) < 0 . 0001 ; p ( WT 4D9 vs APP 4D9 ) < 0 . 0001 ; p ( APP isotype vs APP 4D9 ) = 0 . 9986 ; n . s . , not significant . ( E ) Confocal images of P2RY12 ( red ) , IBA1 ( green ) , and Aβ ( gray ) costainings from cortex . Top panel : Dotted white boxes indicate the area in P2RY12 and IBA1 costainings that are magnified as inset . Bottom panel : Insets show P2RY12 , IBA1 and Aβ null at a larger magnification . Of note , we did not observe a complete co - localization of P2RY12 and null suggesting different microglial populations . Scale bar = 10µm ; scale bar ( insets ) = 5µm . ( F ) Quantification of P2RY12 stained cells in the cortex shown in E . Two - way ANOVA , Tukey ' s multiple comparison test ( Genotype effect : F1 , 23 = 71 . 99 , p = < 0 . 0001 ; Treatment effect : F1 , 23 = 9 . 029 , p = 0 . 0063 ; Genotype x Treatment effect : F1 , 23 = 5 . 93 , p = 0 . 0231 ) ; p ( WT isotype vs APP isotype ) = 0 . 0018 ; p ( WT null vs APP 4D9 ) < 0 . 0001 ; p ( APP isotype vs APP 4D9 ) = 0 . 0051 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Immunohistochemical costainings of TREM2 (magenta) and IBA1 (green) microglia in the cortex of isotype control and 4D9 injected WT and APP-NL-G-F mice. Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes. Scale bar = 10µm; scale bar (inset) = 2.5µm.(C) Quantification of cortical TREM2 stainings shown in B. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,23=70.63, p=<0.0001; Treatment effect: F1,23=14.33, p=0.0010; Genotype x Treatment effect: F1,23=14.51, p=0.0009); p (WT isotype vs WT 4D9) > 0.9999; p (WT isotype vs APP isotype) = 0.02; p (WT 4D9 vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.0001; n.s., not significant.(D) Quantification of cortical IBA1 staining shown in B. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,21=215.4, p=<0.0001; Treatment effect: F1,21=0.5994, p=0.4475; Genotype x Treatment effect: F1,21=0.2992, p=0.5902); p (WT isotype vs APP isotype) < 0.0001; p (WT 4D9 vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.9986; n.s., not significant.(E) Confocal images of P2RY12 (red), IBA1 (green), and Aβ (gray) costainings from cortex. Top panel: Dotted white boxes indicate the area in P2RY12 and IBA1 costainings that are magnified as inset. Bottom panel: Insets show P2RY12, IBA1 and Aβ null at a larger magnification. Of note, we did not observe a complete co-localization of P2RY12 and null suggesting different microglial populations. Scale bar= 10µm; scale bar (insets) = 5µm.(F) Quantification of P2RY12 stained cells in the cortex shown in E. Two-way ANOVA, Tukey's multiple comparison test (Genotype effect: F1,23=71.99, p=<0.0001; Treatment effect: F1,23=9.029, p=0.0063; Genotype x Treatment effect: F1,23=5.93, p=0.0231); p (WT isotype vs APP isotype) = 0.0018; p (WT null vs APP 4D9) < 0.0001; p (APP isotype vs APP 4D9) = 0.0051.


Figure 7 ( A , B ) Overview of the cortex immunohistochemically stained for Aβ plaques in 6 months old APP - NL - G - F mice treated with isotype control ( A ) and 4D9 antibody ( B ) . Scale bar = 100µm . ( C ) Higher magnification images of cortical Aβ plaques in APP isotype and 4D9 injected mice . Scale bar = 10µm . ( D ) Quantification of cortical plaque area from stainings shown in C . Mann - Whitney U test , p = 0 . 0082 . ( E ) Quantification of total plaque coverage of Aβ stainings in the cortex shown in C . Mann - Whitney U test , p = 0 . 014 . ( F ) Immunoblot analysis of soluble ( DEA fraction , top ) and insoluble Aβ levels ( FA fraction , bottom ) in brains of APP - NL - G - F mice treated with either isotype control or 4D9 antibody . While a reduction in levels of soluble Aβ is evident from the DEA fractions no change in levels of insoluble Aβ could be detected in the FA fractions . Note that the higher background in the DEA immunoblot is due to a much longer exposure time , which was needed to visualize soluble Aβ . ( G ) MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in ( F ) . Mann - Whitney U test , p = 0 . 026 . ( H ) MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in ( F ) . Unpaired t - test ( two - tailed ) with Welch ' s correction ; p = 0 . 0877 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A, B) Overview of the cortex immunohistochemically stained for Aβ plaques in 6 months old APP-NL-G-F mice treated with isotype control (A) and 4D9 antibody (B). Scale bar= 100µm.(C) Higher magnification images of cortical Aβ plaques in APP isotype and 4D9 injected mice. Scale bar= 10µm. (D) Quantification of cortical plaque area from stainings shown in C. Mann-Whitney U test, p=0.0082. (E) Quantification of total plaque coverage of Aβ stainings in the cortex shown in C. Mann-Whitney U test, p=0.014.(F) Immunoblot analysis of soluble (DEA fraction, top) and insoluble Aβ levels (FA fraction, bottom) in brains of APP-NL-G-F mice treated with either isotype control or 4D9 antibody. While a reduction in levels of soluble Aβ is evident from the DEA fractions no change in levels of insoluble Aβ could be detected in the FA fractions. Note that the higher background in the DEA immunoblot is due to a much longer exposure time, which was needed to visualize soluble Aβ.(G) MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in (F). Mann-Whitney U test, p=0.026. (H) MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in (F). Unpaired t-test (two-tailed) with Welch's correction; p=0.0877.


Figure 1 ( B ) Maximum intensity projections ( top ) and representative sections ( bottom ) of typical nuclei of the indicated cell types stained with DAPI . Scale bars , 3 μm . ( C ) Areas of DAPI - dense regions ( top ) , distance of DAPI - dense regions from the nuclear periphery ( middle ) , and variance of DAPI signals ( bottom ) . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of DAPI dense regions = 950 / 1450 / 839 / 1535 / 736 and number of slices = 90 / 115 / 95 / 135 / 110 for mESC / EpiLC / d2 / d4c7 mPGCLC / GSC . Significances are computed using Wilcoxon rank - sum tests , p - values from top to bottom 4 . 37e - 3 , 1 . 62e - 3 , 2 . 99e - 2 , 2 . 03e - 10 , 4 . 03e - 1 , < 2 . 2e - 16 , 1 . 31e - 3 , 8 . 94e - 3 , 1 . 06e - 4 , 5 . 62e - 2 , 4 . 63e - 5 , 7 . 65e - 13 . P - value symbol brackets : ∗ ∗ ∗ ∗ = [ 0 , 0 . 0001 ) ; ∗ ∗ ∗ = [ 0 . 0001 , 0 . 001 ] ; ∗ ∗ = [ 0 . 001 , 0 . 01 ) ; ∗ = [ 0 . 01 , 0 . 05 ) ; ns = [ 0 . 05 , ( D ) ( left ) Fluorescence in situ hybridization ( FISH ) against chromosome 16 ( red ) with DAPI staining ( grey ) . Z - stacked representative images are paired with magnified views . ( right ) Distributions of surface volumes for chr16 . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 51 / 68 / 53 for mESC / EpiLC / GSC . Scale bars , 5 μm . Significances are computed using Wilcoxon rank - sum tests , p - values from left to right : 4 . 16e - 2 , 4 . 33e - 6 , 8 . 68e - 9 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei of the indicated cell types stained with DAPI. Scale bars, 3 μm.(C) Areas of DAPI-dense regions (top), distance of DAPI-dense regions from the nuclear periphery (middle), and variance of DAPI signals (bottom). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI dense regions = 950/1450/839/1535/736 and number of slices = 90/115/95/135/110 for mESC/EpiLC/d2/d4c7 mPGCLC/GSC. Significances are computed using Wilcoxon rank-sum tests, p-values from top to bottom 4.37e-3, 1.62e-3, 2.99e-2, 2.03e-10, 4.03e-1, <2.2e-16, 1.31e-3, 8.94e-3, 1.06e-4, 5.62e-2, 4.63e-5, 7.65e-13. P-value symbol brackets: ∗∗∗∗ = [0, 0.0001); ∗∗∗ = [0.0001, 0.001]; ∗∗ = [0.001, 0.01); ∗ = [0.01, 0.05); ns = [0.05,(D) (left) Fluorescence in situ hybridization (FISH) against chromosome 16 (red) with DAPI staining (grey). Z-stacked representative images are paired with magnified views. (right) Distributions of surface volumes for chr16. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 51/68/53 for mESC/EpiLC/GSC. Scale bars, 5 μm. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 4.16e-2, 4.33e-6, 8.68e-9.


Figure 4 ( H ) Western blot for lamin B1 in different cell types ( bottom ) and quantification normalized by β - actin ( top ) . ( L ) ( top ) Representative images of FISH against major satellite repeats in EpiLCs and GSCs . Scale bars , 10 μm ; ( bottom ) percentage of the pericentromeres detached from the nuclear lamina in EpiLCs and GSCs . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 18 / 22 for EpiLC / GSC .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(H) Western blot for lamin B1 in different cell types (bottom) and quantification normalized by β-actin (top).(L) (top) Representative images of FISH against major satellite repeats in EpiLCs and GSCs. Scale bars, 10 μm; (bottom) percentage of the pericentromeres detached from the nuclear lamina in EpiLCs and GSCs. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 18/22 for EpiLC/GSC.


Figure 5 ( I ) ( left ) Western blot for G9a , GLP , Setdb1 and α - tubulin ; ( right ) quantification normalized by α - Tubulin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(I) (left) Western blot for G9a, GLP, Setdb1 and α-tubulin; (right) quantification normalized by α-Tubulin.


Figure 6 ( K ) ( top ) FISH against the Y chromosome ; ( bottom ) sphericity of the Y chromosome FISH signals ; ( right ) distributions Y chromosome surface volumes . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of cells = 89 / 76 / 69 for mESC / EpiLC / GSC . Scale bar , 10 μm . Significances are computed using Wilcoxon rank - sum tests , p - values

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(K) (top) FISH against the Y chromosome; (bottom) sphericity of the Y chromosome FISH signals; (right) distributions Y chromosome surface volumes. The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of cells = 89/76/69 for mESC/EpiLC/GSC. Scale bar, 10 μm. Significances are computed using Wilcoxon rank-sum tests, p-values from


Figure 7 ( B ) Maximum intensity projections ( top ) and representative sections ( bottom ) of typical nuclei of GSCs and GSCLCs stained with DAPI . Scale bars , 3 μm . ( C ) Areas of DAPI - dense regions ( left ) , distance of DAPI - dense regions from the nuclear periphery ( middle ) , and variance of DAPI signals ( right ) . The point marks the median while the thick and thin lines correspond to 66 % and 95 % intervals , respectively . Number of DAPI dense regions = 1535 / 736 / 1227 and number of slices = 135 / 110 / 120 for d4c7 mPGCLC / GSC / GSCLC . Significances are computed using Wilcoxon rank - sum tests , p - values from left to right : 2 . 03e - 10 , 1 . 69e - 9 , 0 . 123 , 0 . 00894 , 8 . 02e - 8 , 0 . 0707 , 7 . 65e - 13 , 0 . 417 , 2 . 07e - 12 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) Maximum intensity projections (top) and representative sections (bottom) of typical nuclei of GSCs and GSCLCs stained with DAPI. Scale bars, 3 μm. (C) Areas of DAPI-dense regions (left), distance of DAPI-dense regions from the nuclear periphery (middle), and variance of DAPI signals (right). The point marks the median while the thick and thin lines correspond to 66% and 95% intervals, respectively. Number of DAPI dense regions = 1535/736/1227 and number of slices = 135/110/120 for d4c7 mPGCLC/GSC/GSCLC. Significances are computed using Wilcoxon rank-sum tests, p-values from left to right: 2.03e-10, 1.69e-9, 0.123, 0.00894, 8.02e-8, 0.0707, 7.65e-13, 0.417, 2.07e-12.


Figure 1D , Scoring of the PI3Kα activation transcriptomic signature was used to cluster patients with high , medium ( both groups were combined , high + medium : red ) and low ( black ) scoring levels in the primary tumours of confirmed PDAC patients from PAAD ( TCGA ) or PACA - AU ( BH corrected p - values ) . The survival curves of each cluster were then plotted , and the statistical significance was calculated using the logrank test .

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Figure 1D, Scoring of the PI3Kα activation transcriptomic signature was used to cluster patients with high, medium (both groups were combined, high+medium: red) and low (black) scoring levels in the primary tumours of confirmed PDAC patients from PAAD (TCGA) or PACA-AU (BH corrected p-values). The survival curves of each cluster were then plotted, and the statistical significance was calculated using the logrank test.


Figure 2E , Murine pancreatic tumour cells R211 were treated with the vehicle , α , β , β / δ , γ - specific or pan - PI3K inhibitors at 0 . 1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . Representative image of filter after Crystal violet staining is shown . Scale = 200µm . n = 3 in each group . F , Murine pancreatic tumour cells R211 were treated with vehicle , BYL - 719 1 µM , AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 4 in each group . G L , PANC - 1 - transduced cells were subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 5 in each group .

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Figure 2E, Murine pancreatic tumour cells R211 were treated with the vehicle, α, β, β/δ, γ-specific or pan-PI3K inhibitors at 0.1 or 1 µM and simultaneously subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. Representative image of filter after Crystal violet staining is shown. Scale=200µm. n = 3 in each group. F, Murine pancreatic tumour cells R211 were treated with vehicle, BYL-719 1 µM, AZD4547 2 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 4 in each group. G L, PANC-1-transduced cells were subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n = 5 in each group.


Figure 3A - B A , Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle , α - specific A66 or pan - PI3K inhibitor BKM120 at 0 . 01 , 0 . 05 , 0 . 1 , 0 . 5 , 1 or 5 µM in the presence of 10 % FBS and protein levels of P - Akt ( Ser473 ) , total Akt and β Actin were observed by western blot . B , P - Akt on Ser473 was quantified and normalised with β Actin . n = 3 in each group . R211 cells were treated with the vehicle , α - specific inhibitor A66 or pan - PI3K inhibitor BKM120 at 1 µM D , migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle ( DMSO ) . n = 3 in each group . E , Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle ( 0 . 01 % DMSO ) or α - specific A66 or pan - PI3K inhibitor BKM120 at 1µM . Phospholipids were extracted , total PIP , PIP2 and PIP3 were quantified and compared to the vehicle ( DMSO ) . n = 3 in each group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A-B A, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle, α-specific A66 or pan-PI3K inhibitor BKM120 at 0.01, 0.05, 0.1, 0.5, 1 or 5 µM in the presence of 10% FBS and protein levels of P-Akt (Ser473), total Akt and β Actin were observed by western blot. B, P-Akt on Ser473 was quantified and normalised with β Actin. n = 3 in each group.R211 cells were treated with the vehicle, α-specific inhibitor A66 or pan-PI3K inhibitor BKM120 at 1 µM D, migrating cells were quantified after 24h in a Boyden chamber assay and compared to vehicle (DMSO). n = 3 in each group.E, Murine pancreatic tumour cells R211 were treated for 15 min with the vehicle (0.01% DMSO) or α-specific A66 or pan-PI3K inhibitor BKM120 at 1µM. Phospholipids were extracted, total PIP, PIP2 and PIP3 were quantified and compared to the vehicle (DMSO). n = 3 in each group.


Figure 4A , Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells ( R211 , PDAC8661 , PANC - 1 , 10593 , 10158 , R6344 , R6430 , R6065 , R6141 ) . The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor . IC50 is determined on recombinant proteins . Pearson correlation tests were performed and its p - values were presented separately for each class I PI3K isoform .

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Figure 4A, Migration inhibition capacities of PI3K inhibitors at 1 µM were tested on pancreatic tumour cells (R211, PDAC8661, PANC-1, 10593, 10158, R6344, R6430, R6065, R6141). The mean of these values was plotted against the in vitro IC50 for each class I PI3K and each inhibitor. IC50 is determined on recombinant proteins. Pearson correlation tests were performed and its p-values were presented separately for each class I PI3K isoform.


Figure 5A , Representative images of PI3K activation ( as shown by positive pAkt substrate staining ) on the primary tumours of PDAC patients , with values of quantification of cfDNA and mutated KRAS allele frequency . C , Survival curve of KPC mice presenting different levels of cfDNA . n in each group is indicated . Log rank test , * * * p < 0 .

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Figure 5A, Representative images of PI3K activation (as shown by positive pAkt substrate staining) on the primary tumours of PDAC patients, with values of quantification of cfDNA and mutated KRAS allele frequency.C, Survival curve of KPC mice presenting different levels of cfDNA. n in each group is indicated. Log rank test, *** p<0.001


Figure 6G , Survival curve of KPC mice treated with the vehicle or BYL - 719 . Log rank test , * p < 0 . 05 . H - J H , Representative images and i , quantification of Ki67 positive cells in pancreatic tumours and in J , metastasized liver sections after treatment with oral doses of vehicle ( 0 . 5 % methyl cellulose with 0 . 2 % Tween - 80 ) or of BYL - 719 ( 50mg / kg ) . K - L K , Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle ( n is indicated ) , quantification at two time points and L , representative images of luminescence via IVIS ® Spectrum in vivo imaging system . N , Tail vein injection experiment in C57 / B6 mice of murine syngeneic pancreatic cancer cells ( Kras mutated A338 , Kras mutated and half PI3Kα inactive , A260 ; n > 8 in each group ) , quantification of micro - and macro - metastasis at final time point .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6G, Survival curve of KPC mice treated with the vehicle or BYL-719. Log rank test, * p<0.05.H-J H, Representative images and i, quantification of Ki67 positive cells in pancreatic tumours and in J, metastasized liver sections after treatment with oral doses of vehicle (0.5% methyl cellulose with 0.2% Tween-80) or of BYL-719 (50mg/kg).K-L K, Treatment regimen of the tail vein injection experiment in nude mice treated with the PI3Kα inhibitor or vehicle (n is indicated), quantification at two time points and L, representative images of luminescence via IVIS® Spectrum in vivo imaging system.N, Tail vein injection experiment in C57/B6 mice of murine syngeneic pancreatic cancer cells (Kras mutated A338, Kras mutated and half PI3Kα inactive, A260; n>8 in each group), quantification of micro- and macro-metastasis at final time point.


Figure 7A - D Blood count of A , white blood cells , B , monocytes , C , granulocytes and D , lymphocytes in KPC mice , with cohort size in each group . h - jH - J H , Representative images and quantification of I , F4 / 80 positive cells and J , CD206 positive cells after the pharmacological inhibition of PI3Kα . Black arrowheads show positive immune cells . S , Murine pancreatic tumour cells R211 were treated with vehicle , TNF - α 20 ng / mL , BYL - 719 1 µM or both treatments and subjected to a Boyden chamber migration assay . Migrating cells were quantified after 24h . n = 4 independent values .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-D Blood count of A, white blood cells, B, monocytes, C, granulocytes and D, lymphocytes in KPC mice, with cohort size in each group.h-jH-J H, Representative images and quantification of I, F4/80 positive cells and J, CD206 positive cells after the pharmacological inhibition of PI3Kα. Black arrowheads show positive immune cells.S, Murine pancreatic tumour cells R211 were treated with vehicle, TNF-α 20 ng/mL, BYL-719 1 µM or both treatments and subjected to a Boyden chamber migration assay. Migrating cells were quantified after 24h. n= 4 independent values.


Figure 1Expression pattern of Rgcc in the embryonic day 14 . 5 ( E14 . 5 ) mouse cortex as detected by in situ hybridization . The right panels showed the expression patterns of NSC markers , including Pax6 , Sox2 , and Ki67 , along the ventricular zone ( VZ ) of the E14 . 5 mouse cortex . Scale bar : 20 μm .

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Figure 1Expression pattern of Rgcc in the embryonic day 14.5 (E14.5) mouse cortex as detected by in situ hybridization. The right panels showed the expression patterns of NSC markers, including Pax6, Sox2, and Ki67, along the ventricular zone (VZ) of the E14.5 mouse cortex. Scale bar: 20 μm.


Figure 2Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex 1 day post IUE . ShRNA targeting Rgcc and control scramble - shRNA were nucleofected separately . Ki67 were co - stained with GFP . To quantify the ratio between NSC markers and GFP positive cells , only the cells in VZ and SVZ are taken into account . And ROIs of 800 * 1900 pixel were randomly picked for counting . Scale bars : 20 μm . Quantification of the relative ratio of Ki67 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex 1 day post IUE . Sox2 were co - stained with GFP . Scale bars : 20 μm . Quantification of the relative ratio of Sox2 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex 1 day post IUE . Pax6 were co - stained with GFP . Scale bars : 20 μm . Quantification of the relative ratio of Pax6 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 5 days post IUE . ShRNA targeting Rgcc and control scramble - shRNA were transfected separately at E12 . 5 . All the brains were harvest at E17 . 5 . Tbr1 were co - stained with GFP . And ROIs of 300 * 300 pixel were randomly picked along Tbr1 positive region for counting . Scale bars : 100 μm . Quantification of the relative ratio of Tbr1 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 4 days post IUE . ShRNA targeting Rgcc and control scramble - shRNA were transfected separately at E13 . 5 . All the brains were harvest at E17 . 5 . Ctip2 were co - stained with GFP . And ROIs of 300 * 300 pixel were randomly picked along Ctip2 positive region for counting . Scale bars : 100 μm . Quantification of the relative ratio of Ctip2 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 4 days post IUE . ShRNA targeting Rgcc and control scramble - shRNA were transfected separately at E13 . 5 . All the brains were harvest at E17 . 5 . Satb2 were co - stained with GFP . And ROIs of 300 * 300 pixel were randomly picked along ventricle for counting . Scale bars : 100 μm . Quantification of the relative ratio of Satb2 and GFP double positive cells versus GFP positive cells . Immunofluorescent staining of coronal sections of the E17 . 5 mouse cortex 4 days post IUE . Scale bars : 100 μm . The ratio of GFP positive cells in VZ / SVZ , IZ , and CP were counted for migration analysis .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. Ki67 were co-stained with GFP. To quantify the ratio between NSC markers and GFP positive cells, only the cells in VZ and SVZ are taken into account. And ROIs of 800*1900 pixel were randomly picked for counting. Scale bars: 20 μm. Quantification of the relative ratio of Ki67 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Sox2 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Sox2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E14.5 mouse cortex 1 day post IUE. Pax6 were co-stained with GFP. Scale bars: 20 μm. Quantification of the relative ratio of Pax6 and GFP double positive cells versus GFP positive cells.Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 5 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E12.5. All the brains were harvest at E17.5. Tbr1 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Tbr1 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Tbr1 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Ctip2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along Ctip2 positive region for counting. Scale bars: 100 μm. Quantification of the relative ratio of Ctip2 and GFP double positive cells versus GFP positive cells. Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. ShRNA targeting Rgcc and control scramble-shRNA were transfected separately at E13.5. All the brains were harvest at E17.5. Satb2 were co-stained with GFP. And ROIs of 300*300 pixel were randomly picked along ventricle for counting. Scale bars: 100 μm. Quantification of the relative ratio of Satb2 and GFP double positive cells versus GFP positive cells.Immunofluorescent staining of coronal sections of the E17.5 mouse cortex 4 days post IUE. Scale bars: 100 μm. The ratio of GFP positive cells in VZ/SVZ, IZ, and CP were counted for migration analysis.


Figure 3Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with SOX2 . Scale bar : 200 μm Quantification of the VZ - like SOX2 - positive rosette area . Each plot represented an individual rosette . H9 , n = 19 ; # 6 , n = 24 ; # 23 , n = 19 from 2 independent experiments . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with PAX6 and Phospho - Histone 3 ( PH3 ) . Scale bar : 200 μm Quantification of the ratio of PH3 and PAX6 double positive cells versus the total number of PAX6 positive cells of each rosette . Each plot represented an individual organoid ( H9 , n = 6 ; # 6 , n = 8 ; # 23 , n = 8 from 2 independent experiments ) . Three to five rosettes were analyzed for each organoid . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with Ki67 . Scale bar : 200 μm . Quantification of the VZ - like Ki67 positive rosette area . Each plot represented an individual rosette . H9 , n = 60 ; # 6 , n = 54 ; # 23 , n = 96 from 2 independent experiments . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with TUJ1 and PAX6 . Scale bar : 200 μm . Quantification of the ratio of TUJ1 area versus PAX6 area . ( H9 , n = 15 ; # 6 , n = 6 ; # 23 , n = 9 from 2 independent experiment ) . Each plot represented an individual organoid . Three to five rosettes were analyzed for each organoid . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with TBR1 and PAX6 . Scale bar : 200 μm . Quantification of the ratio of TBR1 - positive area around PAX6 - positive area versus PAX6 - positive area in each rosette . ( H9 , n = 10 ; # 6 , n = 10 ; # 23 , n = 7 from 2 independent experiment ) . Each plot represented an individual organoid . Three to five rosettes were analyzed for each organoid . Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC - KO hCOs . Sections were stained with CTIP2 and PAX6 . Scale bar : 200 μm . Quantification of the ratio of CTIP2 area versus PAX6 area . ( H9 , n = 8 ; # 6 , n = 8 ; # 23 , n = 8 from 2 independent experiment ) . Each plot represented an individual organoid . Three to five rosettes were analyzed for each organoid .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with SOX2. Scale bar: 200 μm Quantification of the VZ-like SOX2-positive rosette area. Each plot represented an individual rosette. H9, n=19; #6, n=24; #23, n=19 from 2 independent experiments. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with PAX6 and Phospho-Histone 3 (PH3). Scale bar: 200 μm Quantification of the ratio of PH3 and PAX6 double positive cells versus the total number of PAX6 positive cells of each rosette. Each plot represented an individual organoid (H9, n=6; #6, n=8; #23, n=8 from 2 independent experiments). Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with Ki67. Scale bar: 200 μm. Quantification of the VZ-like Ki67 positive rosette area. Each plot represented an individual rosette. H9, n=60; #6, n=54; #23, n=96 from 2 independent experiments.Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TUJ1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TUJ1 area versus PAX6 area. (H9, n=15; #6, n=6; #23, n=9 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with TBR1 and PAX6. Scale bar: 200 μm. Quantification of the ratio of TBR1-positive area around PAX6-positive area versus PAX6-positive area in each rosette. (H9, n=10; #6, n=10; #23, n=7 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid. Immunofluorescent staining were performed on sections of day 45 wildtype H9 and RGCC-KO hCOs. Sections were stained with CTIP2 and PAX6. Scale bar: 200 μm. Quantification of the ratio of CTIP2 area versus PAX6 area. (H9, n=8; #6, n=8; #23, n=8 from 2 independent experiment). Each plot represented an individual organoid. Three to five rosettes were analyzed for each organoid.


Figure 5Immunofluorescent staining of coronal sections of the E14 . 5 mouse cortex one - day post IUE . ShRNA targeting Rgcc and control scramble - shRNA were nucleofected separately . PH3 were co - stained with GFP . The punctuated PH3 cells were at G2 - phase , while PH3 cells with continuous pattern were at M - phase . To quantify the ratio between PH3 and GFP double positive cells and GFP cells , only the cells in VZ and SVZ are taken into account . Scale bar : 20 μm . n ≥ 3 mouse brains for each group . Data are presented as mean ± SEM ; n ≥ 3 mouse brains for each group ; Statistical significance was tested by Student ' s t - test . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Representative pictures of mitotic NSC marked by P - Vimentin and PH3 revealed by DAPI at the apical surface of the E14 . 5 mouse cortex after IUE . A representative vertically dividing cell was shown in upper panel , and a representative horizontally dividing cell was shown in lower panel . White dash line indicates the apical surface and splitting plane . Scale bar : 5 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Immunofluorescent staining of coronal sections of the E14.5 mouse cortex one-day post IUE. ShRNA targeting Rgcc and control scramble-shRNA were nucleofected separately. PH3 were co-stained with GFP. The punctuated PH3 cells were at G2-phase, while PH3 cells with continuous pattern were at M-phase. To quantify the ratio between PH3 and GFP double positive cells and GFP cells, only the cells in VZ and SVZ are taken into account. Scale bar: 20 μm. n≥3 mouse brains for each group. Data are presented as mean ± SEM; n≥3 mouse brains for each group; Statistical significance was tested by Student's t-test. **, p<0.01; ***, p<0.001;Representative pictures of mitotic NSC marked by P-Vimentin and PH3 revealed by DAPI at the apical surface of the E14.5 mouse cortex after IUE. A representative vertically dividing cell was shown in upper panel, and a representative horizontally dividing cell was shown in lower panel. White dash line indicates the apical surface and splitting plane. Scale bar: 5 μm.