EMBO/SourceData huggingface dataset:
Figure 6A Quantification of TMRM fluorescence by flow cytometry in U2OS:GFP‐parkin cells treated with 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), and 20 μM CCCP (CCCP) for the indicated time, represented as a fraction of fluorescence intensity compared to DMSO‐treated cells. Error bars represent the mean ± s.e.m. (n = 3 experiments).B U2OS:GFP‐parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for TOM20 (red). Scale bars, 50 μm.C Quantification ofPDHE2/E3 bp−positive/TOM20−negative structures from (B); both the total number (white bars) and the number colocalizing with GFP−parkin (gray bars) are indicated. Error bars represent the mean ± s.e.m. P−values are given first for GFP−/GFP−parkin−positive vesicles, then for total vesicle number (n = 42-60 cells in three experiments); ns, not significant; ***P < 0.001).D U2OS:GFP−parkin cells were treated with DMSO (DMSO), 25 μM antimycin A (anti A), 25 μM antimycin A and 10 μM oligomycin (anti A + oligo), or 20 μM CCCP (CCCP) for two hours, then fixed and immunostained for PDH E2/E3 bp (red) and TOM20 (blue). A merged image of both mitochondrial markers (mito) is shown at the far right. Arrows indicate PDH E2/E3 bp−positive/TOM20−negative structures colocalizing with GFP−parkin (green). Scale bars, 20 μm.
Figure 7A Representative immunoblot of whole‐cell lysates from U2OS:GFP and GFP‐parkin cells treated with DMSO, 25 μM antimycin A (anti A), 25 μM antimycin A with 10 μM oligomycin (anti A + oligo), or 20 μM CCCP for the indicated time period.B Quantification of PDH E1a signal intensity relative to that of actin in immunoblots from (A). Error bars represent the mean ± s.e.m. (n = 3 experiments).C Quantification of mitochondrial clearance in U2OS:GFP−parkin cells treated as in (A), fixed and immunostained for TOM20. Data are shown as percentage of cells containing mitochondria, by TOM20 staining, visualized by fluorescence microscopy. Error bars represent the mean ± s.e.m. (n = 3 experiments, with at least 85 cells quantified per condition, per experiment).D U2OS:GFP‐parkin cells were treated as in (A) for the indicated time period, then fixed and immunostained for TOM20 (red). Cell boundaries are delineated in single‐channel images. Scale bars, 20 μm.
Figure 1(A) Representative photomicrographs of MAP2 (green) and cleaved caspase-3 (red) immunocytochemistry of embryonic cortical neurons from wild type (upper panels) or C259A mice treated with vehicle or 10µM Aβ oligomers for 14h. Scale bar 15µm.(B) Quantification of active (cleaved) caspase-3 in cultured cortical neurons of the indicated genotypes treated for 14h with vehicle or Aβ oligomers (2.5, 5, or 10 µM). Data are expressed as percentage cleaved caspase-3 and MAP2 double positive cells over the total number of MAP2 positive cells.(C) Quantification of neurite length in in cultured cortical neurons of the indicated genotypes treated for 24h with vehicle or Aβ oligomers (2.5, or 10 µM). Data are expressed as mean neurite length in individual neurons, relative to wild type vehicle (set to 100 units).
Figure 2(A) Immunohistochemistry for p75NTR in hippocampal CA1 of 5 month wild type, 5xFAD and p75NTR knock-out (KO) mice. Scale bar, 20μm.Western blot analysis of p75NTR expression in total lysates of hippocampus from 6 month old wild type (WT) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain The lower panel shows reprobing for GAPDH to control for equal gel loading. (C) Quantification of p75NTR expression in mouse hippocampus relative to GAPDH.(D) Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice of the indicated ages. Scale bar, 400μm. (E) Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Aβ plaques (mean ± SEM, N=5 mice per group). *, P<0.05 and **, P<0.01 vs 5XFAD. #, P<0.05 vs 5XFAD/KO (one-way ANOVA followed by post hoc test). (F) Quantification of the number of Aβ plaques larger than 30um in diameter per um2 in coronal sections through the hippocampus of 9 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Color codes are as in panel (E). Histogram shows mean ± SEM, N=4 mice per group; *, P<0.05 KO vs. knock-in genotypes. ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ monomers (G) refers to the soluble fraction after Tris-buffered saline extractionELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ oligomers (H) to the soluble fraction after RIPA buffer extraction of the Tris-buffered saline pellet,ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants Aβ fibrils (I) to the soluble fraction after formic acid treatment of the RIPA pellet.
Figure 3(A) Immunostaining for Glial fibrillary acidic protein (GFAP), a marker of astrocytes, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 50μm. (B) Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining (mean ± SEM, N=5 mice per group). (C) Immunostaining for Ionized calcium binding adaptor molecule 1 (Iba1), a marker of microglial cells, and Aβ plaques in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A. Scale bar, 300μm. Right hand panels show high magnification of the indicated areas. Scale bar, 10μm. (D) Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining (mean ± SEM, N=5 mice per group). (E) Immunostaining of reticulon 3 (RTN3), a marker of dystrophic neurites, and Aβ plaques in coronal sections through the hippocampus of 6 month old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 40μm. (F) Quantification of RTN3-positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining (mean ± SEM, N=5 mice per group).(G) MitoSox staining, a mitochondrial superoxide indicator, and DAPI in coronal sections through the hippocampus of 6 month old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A mice. Scale bar, 60μm. (H) Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows MitoSox mean fluorescence intensity in arbitrary units (mean ± SEM, N=5 mice per group).
Figure 4(A) Percentage of change in field excitatory postsynaptic potential (fEPSP) recorded after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from 6 month old wild type, 5xFAD and 5xFAD/p75NTR mutant mice, as indicated. Results are presented as mean % change normalized to t=0 ± SEM. N=6 (wild type), 5 (5xFAD), 9 (5xFAD/KO), 7 (5xFAD/ΔDD) and 8 (5xFAD/C259A) slices from 3 mice per genotype, respectively. (B) Quantification of fEPSP (mean % change ± SEM) in the indicated genotypes at 3 time points. *, P<0.05; **, P<0.01 vs. 5xFAD (two-way ANOVA followed by post hoc test). N numbers as in (A).(C) Behavior in the novel object recognition (NOR) test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean recognition index ± SEM during training, and 30min, 24h and 14 days after training, corresponding to measures of short term, long term and remote memory, respectively. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD (two-way ANOVA followed by post hoc test). N=12 (wild type, 5xFAD/ΔDD and 5xFAD/C259A), 10 (5xFAD) and 8 (5xFAD/KO) mice per genotype, respectively.(D) Training latency in the Barnes maze test of 6 month old wild type, 5XFAD and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions. Bar color codes are as in panel (A). *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two-way ANOVA followed by post hoc test). N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively.N=14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, (5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively. (E) Percentage of time spent (mean ± SEM) in the target quadrant of the Barnes maze test 30min, 24h and 14 days after training. *, P<0.05 vs. 5xFAD; #, P<0.05 vs. wild type, 5xFAD/ΔDD or 5xFAD/C259A (two way ANOVA followed by post hoc test).
Figure 5(A) Western blot analysis of CTF beta (CTFβ) in hippocampal lysates of 9 month old 5xFAD mice carrying different p75NTR alleles detected using anti-APP-CTF antibody Arrows point to phospho-C99 and C99 CTFβ species Asterisk denotes different species of native and phosphorylated alpha and beta CTFs of lower molecular weights Lower panel shows reprobing for GAPDH. (B) Quantification (mean ± SEM) of phospho-C99/C99 CTFβ species, normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs, 5xFAD/KO; ***, P<0.001 5xFAD vs. 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P=0.056 5xFAD/ΔDD vs. 5xFAD/KO. N=5 mice per group.(C) Western blot analysis of soluble APP alpha (sAPPα) in the soluble Tris-buffered saline fraction of hippocampal homogenate made from 9 moth old 5xFAD mice carrying different p75NTR alleles detected using 6E10 antibody Lower panel shows reprobing for GAPDH. (D) Quantification (mean ± SEM) normalized to GAPDH and expressed relative to levels in 5xFAD mice. *, P<0.05 5xFAD vs. 5xFAD/KO, 5xFAD/ΔDD and 5xFAD/C259A; #, P<0.05 5xFAD/C259A vs. 5xFAD/KO; §, P<0.05 5xFAD/ΔDD vs. 5xFAD/KO. N=9 mice per group.
Figure 6(A) Internalization of triple mutant hAPP in wild type, p75NTR knock-out (KO), ΔDD and C259A hippocampal neurons (15min time point). Counterstaining for p75NTR and DAPI are also shown. Scale bar, 10μm.(B) Kinetics of internalization of triple mutant hAPP in hippocampal neurons from wild type, p75NTR knock-out (KO), ΔDD and C259A mice. Shown is mean ± SEM of percentage internalization of total surface APP (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in vs. KO mutants; **, P<0.01, WT vs. all other genotypes (2-way ANOVA). (C) Linear transformation of hAPP internalization kinetics shown in (B). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes.(D) Kinetics of internalization of p75NTR in wild type hippocampal neurons in the presence or absence of NGF. Shown is mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 ± NGF (2-way ANOVA). (E) Linear transformation of p75NTR internalization kinetics IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes.(F) Internalization of endogenous p75NTR in wild type hippocampal neurons (30min time point). Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.(G) Kinetics of internalization of p75NTR in hippocampal neurons from wild type, ΔDD and C259A mice. Show are mean ± SEM of percentage internalization of total surface p75NTR (set to 100%). N=3 independent experiments each performed in duplicate; *, P<0.05 knock-in mutants vs. WT (2-way ANOVA). (H) Linear transformation of p75NTR internalization kinetics shown in (G). IMAX denotes maximal internalization in %. T1/2 denotes time for half maximal internalization in minutes. (I) (A) Co-immunoprecipitation between p75NTR and β-adaptin in hippocampal extracts from 6 month wild type and p75NTR mutant mice as indicated. Results shown are representative from 2 independent experiments. Molecular weights are in kDa. IP: immunoprecipitation; IB: immunoblotting; WCL, whole cell lysate.
Figure 7(A) Co-immunoprecipitation between hAPP and p75NTR in hippocampal extracts from 6 month 5xFAD and p75NTR mutant mice as indicated. Results shown are representative from 3 independent experiments. Molecular weights are indicated in kDa. IP: immunoprecipitation; IB: immunoblotting; WCL, whole cell lysate.(B) Proximity ligation assay (PLA) between triple mutant hAPP and p75NTR in hippocampal neurons from wild type and C259A mice (first and second rows, respectively). The third row (no APP) shows control PLA reaction in neurons that did not receive hAPP lentivirus. The fourth row (KO) shows control PLA reaction in neurons from p75NTR knock-out mice infected with hAPP lentivirus. Counterstaining for MAP2 is also shown. Scale bar, 10μm.(C) Quantification of total PLA signals in wild type and p75NTR mutant neurons in the presence or absence of hAPP lentivirus, as indicated. Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments. (D) Quantification of cell surface PLA signals in wild type and p75NTR mutant neurons in the presence or absence of triple mutanthAPP lentivirus, as indicated. Live neuron cultures were fed with anti-mouse p75NTR and anti-hAPP antibodies on ice, washed after staining, fixed, and developed with PLA reaction. Values were normalized to levels in wild type neurons and are expressed as mean PLA puncta per cell ± SEM from at least 25 neurons in 3 independent experiments. (E) Internalization of hAPP/p75NTR PLA signals in hippocampal neurons of wild type and p75NTR mutant mice. Live neuron cultures were fed with anti-mouse p75NTR and anti-hAPP antibodies on ice, washed and plates placed at 37°C for different periods of time to allow internalization. Internalization was stopped by acid wash, followed by fixation and PLA reaction. Counterstaining for MAP2 and DAPI are also shown. Scale bar, 10μm.(F) Kinetics of co-internalization of hAPP and p75NTR in hippocampal neurons from wild type, ΔDD and C259A mice. Show are mean ± SEM of percentage internalization of total surface PLA signal (set to 100%). (G) Linear transformation of p75NTR internalization kinetics
Figure 8(A) Micrographs of triple mutant hAPP after 15min internalization (red), BACE1 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main images. Scale bar, 5μm. (B) Quantification of the proportion of BACE1 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % BACE co-localized with hAPP. N=3 independent experiments, each performed in duplicate; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (C) Quantification of the proportion of internalized hAPP that co-localized with BACE1 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with BACE1. N=3 independent experiments, each performed in duplicate; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test).(D) Micrographs of p75NTR after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (E) Quantification of the proportion of Rab11 that co-localized with p75NTR after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with p75NTR. n=4; *, P<0.05; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (F) Quantification of the proportion of internalized p75NTR that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized p75NTR co-localized with Rab11. n=4; **, P<0.01 vs. WT (one-way ANOVA followed by post hoc test). (G) Micrographs of triple mutant hAPP after 15min internalization (red), Rab11 immunocytochemistry (green) and their superimposition in wild type hippocampal neurons infected with 5xFAD hAPP lentivirus. DAPI and MAP2 staining are shown in the left column. Insets show higher (3.5x) magnification of the areas indicated in the main merged images. Scale bar, 5μm. (H) Quantification of the proportion of Rab11 that co-localized with hAPP after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % Rab11 co-localized with hAPP. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test). (I) Quantification of the proportion of internalized hAPP that co-localized with Rab11 after 6 and 15 min of internalization at 37°C. Results are expressed as mean ± SEM of % internalized hAPP co-localized with Rab11. n=4; *, P<0.05 vs. WT (one-way ANOVA followed by post hoc test).
Figure 1F smFISH of Arglu1 pre-mRNA (magenta) and Arglu1 sisRNA (green). Merged dots (white, arrowheads) represent Arglu1 pre-mRNA transcript. G Quantification of Arglu1 pre-mRNA and Arglu1 sisRNA copy number from (F). (n = 50 cells) Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.I RT-PCR of Arglu1 sisRNA, spliced mRNA levels and actin as control after AMOs treatment.
Figure 2G Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in control cells and Arglu1 sisRNA KD cells. Right: Quantification of relative ARGLU1 protein levels normalized to GADPH in Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).M Left: RT-PCR of AXIN1a and AXIN1b spliced levels and actin as loading control in 3 days Arglu1 sisRNA KD cells vs control cells under 100 nM estrogen treatment for 2 hrs. Right: Quantification of relative spliced AXIN1a/AXIN1b expression ratio normalized to actin from RT-PCR analysis (left). (n = 3 biological replicates).
Figure 3B Left: Western blot analysis of ARGLU1 protein and GAPDH as loading control in 1 day Arglu1 sisRNA KD cells vs control cells. Right: Quantification of relative ARGLU1 protein expression level normalized to GADPH in 1 day Arglu1 sisRNA KD cells vs control cells from western blot analysis (left). (n = 3 biological replicates).D Left: smFISH of Arglu1 sisRNA (green) coupled with immunostaining of ARGLU1 protein (red) shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowheads) of Arglu1 sisRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 sisRNA and ARGLU1 protein in the direction of the white arrow. E Left: smFISH of Arglu1 pre-mRNA (green) coupled with ARGLU1 protein (red) immunostaining shown in z-stack. Right: Zoom in z-slice on the boxed area showing co-localization (arrowhead) of Arglu1 pre-mRNA with ARGLU1 protein. The intensity plot shows the signal quantification and co-localization of Arglu1 pre-mRNA and ARGLU1 protein in the direction of the white arrow.F smFISH of Arglu1 sisRNA (green) and Arglu1 pre-mRNA (magenta) in 1 day Arglu1 sisRNA KD cells vs control cells. G, H Quantification of Arglu1 sisRNA and Arglu1 pre-mRNA copy number from (F). (n = 90 cells). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.
Figure 4A Immunostaining of MED1 (magenta) and SC35 (red) with transiently expressed mCherry-ARGLU1 protein (green) in MCF-7 cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of mCherry-ARGLU1 protein, MED1 and SC35 in the direction of the white arrow.B Confocal images of GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) at the pre-bleach, bleach, and post-bleach time points in FRAP analysis. The boxed area showing the speckle that was being bleached. C Fluorescence intensities of the nuclear speckle over time in GFP-SRSF2 (green, control) and mCherry-ARGLU1 protein (red) FRAP analysis. (n = 3 separate nuclear speckles).D Immunostaining of ARGLU1 protein (green) and SC35 (red) in 1 day Arglu1 sisRNA KD cells vs control cells. Zoom in of boxed area and the intensity plot shows the co-localization and signal quantification of ARGLU1 protein and SC35 in the direction of the white arrow.E Confocal images of 125 nM ARGLU1-GFP protein (green) only and ARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either Arglu1 intron 2 with UCE (Intron 2 WT) or IVT Arglu1 intron without UCE (Intron 2 ΔUCE). White arrowhead indicates ARGLU1-GFP droplet incorporated with IVT Intron 2 WT and blue arrowhead indicates free ARGLU1-GFP droplet not incorporated with IVT Intron 2 WT. F Quantification of total ARGLU1-GFP droplets from (E). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. G Quantification of average ARGLU1-GFP droplets size with/without incorporation of IVT intron 2 WT from (E, ARGLU1-GFP + Intron 2 WT). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.
Figure 6A Western blot analysis of dARGLU1 protein level from dARGLU1 protein immunoprecipitation in S2 cells. Samples with rabbit-IgG antibodies were used as control. ARGLU1 antibody #1, but not #2, worked for IP.F Left: Western blot analysis of dARGLU1 protein levels and ACTIN5C as loading control in dArglu1 sisRNA KD ovaries vs control (sibling control, MTD-GAL4/CyO) ovaries. Right: Quantification of relative dARGLU1 protein levels in dArglu1 sisRNA KD ovaries vs control ovaries from the western blot analysis. (n = 3 biological replicates)G Left: Immunostaining of dARGLU1 protein (green) and VASA (red) in dArglu1 sisRNA KD germaria vs control (sibling control, MTD-GAL4/TM3) germaria. The asterisk (*) marks the anterior of the germarium. Right: Zoom in of the boxed area and the intensity plot shows the signal quantification of dARGLU1 protein and VASA in the direction of the white arrow.Confocal images of 125 nM dARGLU1-GFP protein (green) only and dARGLU1-GFP protein added with 50 nM IVT RNA (magenta), of either dArglu1 intron 2 until the 3' end of sisRNA (dArglu1 intron 2) or IVT dArglu1 intron 2 starting after 3' end of sisRNA (dArglu1 pre-mRNA). White arrowheads indicate dARGLU1-GFP droplets incorporated with IVT dArglu1 intron 2 and blue arrowheads indicate free dARGLU1-GFP droplets not incorporated with IVT dArglu1 intron 2. J Quantification of total dARGLU1-GFP droplets from (I). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. K Quantification of average dARGLU1-GFP droplets size with/without incorporation of IVT dArglu1 intron 2 from (I, dARGLU1-GFP + dArglu1 intron 2). (n = 3 biological replicates). Cross, mean; Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. D
Figure 7A Confocal images of 125 nM d/hARGLU1-GFP protein added with 50 nM IVT RNA of either hArglu1 intron 2 WT (magenta) or dArglu1 intron 2 (red). White arrowheads indicate free IVT RNA dots that are not co-localized with the d/hARGLU1-GFP droplets. B Quantification of non co-localized RNA dots from (A, white arrowheads). (n = 16 biological replicates). C, D Quantification of total hARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates). E, F Quantification of total dARGLU1-GFP droplets and the average droplet size with/without incorporation of IVT hArglu1 intron 2 WT or dArglu1 intron 2 from (A). (n = 3 biological replicates).