EMBO/SourceData huggingface dataset:
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.
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.
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.
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 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.
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.
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.
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.
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.
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.
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.
Figure 6RGCC was co-stained with PCNT on 3D hCO slices. RGCC was with stronger expression in NSCs during M-phase. prometaphase (B'), metaphase (B'') anaphase (B''') and telophase (B''''). RGCC was expressed around nucleus in interphase (B''''') . NSCs undergoing prometaphase, metaphase, anaphase and telophase were pointed out by yellow arrow. Interphase were pointed out by white arrow (B'''''). Immunostaining of 2D-differentiated NSCs in metaphase showed partial colocalization of RGCC with α-TUBULIN in the spindle (B'''''', yellow arrow). Scale bar: 5 μm.The Western blots results. Proteins were collected from WT hCOs and RGCC KO hCOs #6 at day 45. B-ACTIN were used as references.PCNT was co-stained with β -TUBULIN on WT NSCs and RGCC KO NSCs differentiated by 2D cultured dual-SMAD inhibition method. Scale bar: 5μm.
Figure 1(A) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) and PMEPA1 (high and low exposures displayed) in MB patient sample lysates from different groups: WNT (blue), SHH (red), Group 3 (yellow) or Group 4 (green). β-actin was used as a loading control. Relative quantification of P-Smad2 signal to β-actin (P-S2/β-Actin) and total Smad2 (P-S2/Tot-S2) are indicated below the blots.(B) Boxplots summarizing the expression of INHBB, TGFB1 and TGFB3 ligands of the TGFβ/Activin pathway in the different groups of MB (blue WNT, red SHH, yellow Group 3 and green Group 4) and in fetal and adult cerebellum (grey) in the dataset of "Data ref: Cavalli et al, 2017". Wilcoxon rank-sum tests were performed to determine p-values for panel B. p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.(C) Immunoblot analysis of phosphorylated Smad2 (P-Smad2) in non-Group 3 (blue) and Group 3 (yellow) MB cell lines on the left panel. The level of total Smad2 (Smad2) was assessed and β-actin was used as a loading control. On the right panel, relative level of P-Smad2 (P-S2) were quantified to total β-actin. The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.(D) RT-qPCR were performed on RNA extracted from non-Group 3 (blue) and Group 3 (yellow) MB cell lines to compare expression levels of INHBB (left) and TGFB3 (right). The p-values were determined by unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bars represent the mean ± SD. Number of replicates is n≥3.