EMBO/SourceData huggingface dataset:
Figure 3B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. C Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. D Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse. E Transduction rates of AAV9-PHP.B at 8, 16, and 32kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate.F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4.G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured.H 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.
Figure 4A Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4-/-, and Syne4-/- mice injected with AAV.Syne4. B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4-/- mice and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate. C Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm.D Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4-/- mice, 30 IHC and 90 OHC from 2 Syne4-/- mice injected with AAV.Syne4 were measured.
Figure 5A Representative example of ABR traces obtained at 4w from a WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4 in response to an 18 kHz stimulus.B ABR thresholds at 4w of WT, Syne4-/-, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=7 for WT, n=8 for Syne4-/-, n=20 for Syne4-/- + AAV.Syne4, n=6 for Syne4-/- + AAV.GFP. C Quantification of P1-N1 amplitude delta from (B), n=4 for WT, n=4 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4. D Quantification of P1 latency from (B), n=4 for WT, n=2 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4. E DPOAE thresholds at 4w, n=6 for WT, n=5 for Syne4-/-, n=6 for Syne4-/- + AAV.Syne4. F ABR threshold at 12w, n=6 for WT, n=8 for Syne4-/-, n=16 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP.
Figure 7B, C Quantification of activity level and cumulative freezing duration at 12w during seconds 90-210 of day 2 in WT, Syne4-/- mice, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=14 for WT, n=16 for Syne4-/-, n=9 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP. (C) Activity level. (B) Cumulative freezing duration.
Figure 1F-I ChIP assays with antibodies against H3K9me3, H4K20me3, H3K4me3 and H3K9Acetyl were performed as in (D). Only 'C' position signals were showed and normalized to Input.M ChIP assays with antibodies against CDK9 and Pho-Pol II were performed in siNC and siCHAF1A J-Lat 10.6 cells. ChIP signals in each group were normalized to Input.
Figure 2siRNAs targeting each CAF-1 subunits and corresponding enriched genes were transfected into TZM-bl cell lines respectively. The fold change of luciferase expression of each groups was normalized to siNC.
Figure 3C, D ImmunoFISH images of CAF-1 bodies and HIV-1 genomic DNA within J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were acquired by treating with TNFα.E RFP-tagged CHAF1A and GFP-tagged PML body component SP100 were co-overexpressed in HEK293T cells and imaged by SIM. F GFP-tagged CHAF1A and RFP-tagged BRD4 were co-overexpressed in HEK293T cells and imaged by SIM.SUV39H1, HP1α, DNMT1, MBD1, RING1B, EZH2 were co-overexpressed with CHAF1A in HEK293T cells respectively. SIM imaging was performed for each combination.
Figure 4E GFP-tagged CHAF1A-N-NLS and CHAF1A-C were imaged with RFP-tagged CHAF1A respectively. N: CHAF1A N-terminal. NLS: nuclear localization signal of CHAF1A, which was presented within C-terminal. C: CHAF1A C-terminal.F Upper panel showed WT-CHAF1A bodies. Lower panel showed dLLPS-CHAF1A which was CHAF1A-Q34A-I116A-K395D-R402D-K409D.G-I GFP-tagged CHAF1A-IDR and CHAF1A-mIDR were purified in vitro. Ten micromole of each protein was incubated with gradient droplet formation buffer. The droplets of CHAF1A-IDR appeared in NaCl concentration of 125 mM or lower (G). No CHAF1A-mIDR droplet was detected within any NaCl concentration (I). The volumes of CHAF1A-IDR droplets increased with the increase of protein concentration (H). Left subpanels within each panel represented images of protein droplets in different buffers. Right subpanels within each panel represented statistical analysis results of droplets areas and numbers.
Figure 5B First panel: the co-localization of PCNA and CHAF1A. Second panel: the distribution of PCNA in the absence of endogenous CHAF1A. Third panel: the distribution of PCNA in the presence of CHAF1A-dPIP1 mutant and the absence of endogenous CHAF1A. Fourth panel: the co-localization of PCNA and over-expressed DNMT1 in the absence of endogenous CHAF1A.C Upper two panels: the distribution of HP1α in the presence or absence of CHAF1A. Lower two panels: the distribution of HP1α in the presence of CHAF1A-dHP1BD or CHAF1A-DS2-dHP1BD. Both images of lower two panels were in the absence of endogenous CHAF1A.The distribution of SUV39H2 (E) and MBD1 (F) in the presence or absence of CHAF1A.G The distribution of SUMO paralogs in the absence of CHAF1A.
Figure 6A Flow cytometry figures of CAF-1 body rescue experiment. First figure: Wildtype J-Lat 10.6. Second figure: CHAF1A knock-out J-Lat 10.6. Third figure: CHAF1A-KO J-Lat 10.6 was re-introduced with wildtype CHAF1A. Fourth figure: CHAF1A-KO J-Lat 10,6 was re-introduced with dLLPS-CHAF1A which harbored five mutations. The percentages of GFP-positive cells were labeled in the top right corner of each figure.
Figure 7D The amounts of intracellular HIV-1 RNAs of latently infected primary CD4+ T cells which were isolated from PBMCs of HIV-1-infected individuals. αCD3/αCD28/IL-2 treatment was performed as positive control. shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A. shluc lentiviruses treatment was performed as negative control. SAHA was added as LRA supplement.
Figure 2A Representative photographs of fly eyes expressing (CGG)90-EGFP under a GMR-GAL4 driver, with various belle disruptions.B Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with belle disruptions (Mann-Whitney U test with Bonferonni corrections for multiple comparisons; n=35-77/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).Longevity assays of (CGG)90-EGFP; ElaV-GS (n=147-299/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).G Abundance of (CGG)90-EGFP mRNA normalized to RPL32 mRNA, following belle knockdown, determined by qRT-PCR (n=8/genotype).H Western blot of AUG-driven EGFP in EGFP; Tub5-GS flies with and without belle knockdown.I Quantitation of EGFP band density, normalized to β tubulin band density, from blot in H (n=4/genotype).
Figure 3A Dose-response curves showing the effects of two independent anti-DDX3X siRNAs on the expression of AUG-NL-3xF (top) and (CGG)100 +1 NL-3xF (bottom) reporters. Plasmid-based reporters were transfected into HeLa cells 24 hours after knockdown, and reporter expression was quantified by luminescence. Nanoluciferase (NL) luminescence has been normalized to luminescence from firefly luciferase (FF), which was co-transfected, in order to control for transfection variability. Asterisks refer to comparisons between anti-DDX3X siRNAs and siRNAs against EGFP (siEGFP; two-way ANOVA with Dunnett's multiple comparisons test; n=12/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).(CGG)n +1 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).(CGG)n +2 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).Western blots of FMRpolyG-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.Western blots of FMRpolyA-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.
Figure 4A Expression of in vitro transcribed AUG, +1 (CGG)100, and +2 (CGG)100 NL-3xF RNAs following DDX3X knockdown in HeLa cells, expressed as NL luminescence normalized to FF luminescence. (Student's t test with Bonferroni corrections for multiple comparisons; n=21/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).B Abundance of reporter mRNAs following DDX3X knockdown and plasmid-reporter transfection, determined by qRT-PCR. (n=7/condition.) This panel depicts data as means ± SEM.C Expression of AUG-NL-3xF and +1 (CGG)100 NL-3xF in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X (two-way ANOVA with Tukey's multiple comparisons test; n=4/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).D Enrichment of HSPA1A and +1 (CGG)100 NL-3xF mRNA following anti-DDX3X RIP, relative to incubation with isotype control IgG. MALAT RNA, in contrast, is not enriched (Student's t test, n=3).E Expression of +1 and +2 (CGG)100 NL-3xF plasmid reporters with and without an AUG inserted 5' to the CGG repeat, with and without DDX3X knockdown. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing either +1 or +2 (CGG)100 NL-3xF and those expressing the respective AUG-driven variant (two-way ANOVA with Tukey's multiple comparisons test; n=11-12/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).F Expression of NL-3xF plasmids with initiator AUG codons mutated to near-AUG codons, with and without DDX3X knockdown (two-way ANOVA with Dunnett's multiple comparisons test; n=18-24/condition). Black asterisks refer to comparisons between siEGFP-treated and siDDX3X-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter; white asterisks refer to comparisons between siDDX3X-treated cells expressing +1 (CGG)100 NL-3xF and those expressing a different reporter. Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).G Expression of in vitro transcribed near-AUG reporter RNAs in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X. (n=4/group).
Figure 5A Representative photographs of GMR-GAL4; (CGG)90-EGFP fly eyes expressing manipulations of eIF4B and eIF4H. (B) Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with eIF4B/H manipulations (Mann-Whitney U test with Bonferroni corrections for multiple comparisons; n=26-55/genotype). Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).C, D Expression of plasmid-based AUG-NL and +1 (CGG)100 NL-3xF reporters (C), or co-transfected AUG-FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP- and siEIF4B/H-treated cells; pink and blue asterisks refer to comparisons between siEIF4B- (pink) or siEIF4H- (blue) treated cells expressing AUG-NL-3xF and those expressing +1 (CGG)100 (two-way ANOVA with Tukey's multiple comparisons test, n=9/condition).E Expression of plasmid-based AUG-NL-3xF and (CGG)100 +1 NL-3xF reporters with and without over-expression of EIF4B, EIF4H, or both (two-way ANOVA with Dunnett's multiple comparisons test; n=20/condition). Asterisks refer to comparisons between cells over-expressing either EGFP or EIF4B, EIF4H, or EIF4B and EIF4H and expressing the same reporter. Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).
Figure 6A Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF1 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF1-transfected cells; green asterisks refer to comparisons between EIF1-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).B Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF5 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF5-transfected cells; pink asterisks refer to comparisons between EIF5-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).
Figure 7A Sample micrographs collected by automated longitudinal fluorescence microscopy, demonstrating the automated determination of cell death.B Anti-DDX3X western blot of B35 cells transfected with either of two independent anti-DDX3X LNAs or a control LNA.C Expression of EGFP in primary rat neurons transfected with (CGG)100 (+1) EGFP and either anti-DDX3X LNAs or a control (one-way ANOVA with Tukey's multiple comparisons test; n=2408-5689 cells/condition). All graphs depict pooled data, normalized first within the replicate. Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).D, E Transfection of anti-DDX3X LNA #1 (D) or #2 (E) reduced the cumulative risk of death in (CGG)100 (+1) EGFP-expressing neurons (Cox proportional hazard analysis; n=2408-3676 cells/condition). Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).
Figure 1A Immunofluorescence images of MMS22L foci formation at etoposide (ETP)-induced DSBs during S, M and G1 phases of the cell cycle in HeLa cells. Scale bar: 5 μm.B Quantification of a representative experiment from (A) shows number of MMS22L foci per nucleus during indicated cell cycle phases (n = 2; nnuclei ≥ 60).C Immunofluorescence images of CPT-induced MMS22L, RPA2, BRCA2 and RAD51 foci in U2OS cells. Scale bar: 5 μm.D Quantification of a representative experiment (n = 3) from (C) shows percentage of γH2AX, RPA2, BRCA2 and RAD51 foci co-localizing with MMS22L foci (nnuclei = 30; nuclei with ≥ 25 MMS22L foci were analyzed).
Figure 2A Electrophoretic mobility shift assay monitoring MMS22L-TONSL binding to ssDNA without RPA and with RPA-coated ssDNA.B Quantification of data as in (A) shows percent MMS22L-TONSL binding to ssDNA with or without RPA, n = 2; error bars, SEM.C Schematic (left) and western blots (right) of streptavidin and NiNTA pulldowns of his-tagged MMS22L-TONSL, RPA and biotinylated ssDNA.D Yeast two-hybrid analysis shows the effect of deletion of the amino acids 384-522 of MMS22L on the interaction with RPA. The interaction between indicated MMS22L variants fused to LexA DNA-binding domain and RPA1 fused to GAL4 activating domain (GAD) is assessed by survival on media containing 3-aminotriazole (3-AT) in the absence of histidine and by β-galactosidase activity (X-Gal) assays.E Images of MMS22L foci formed in U2OS cells stably expressing wild-type (WT) or Δ384-522 variants of siRNA-resistant HSS-MMS22L, depleted of endogenous MMS22L by RNAi and treated with 50 nM CPT for 18 hours. Scale bar: 5 μm.
Figure 3(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.A Abundance of MMS22L-TONSL at active and HU-stalled replication forks, and fully replicated DNA.(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.B, C MMS22L dynamics at stalled replication forks. Quantification shows average relative abundance of indicated proteins detected in immunoblots, n = 3 except of time-point 5 h, n = 1; error bars, SEM.(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.D, E Effect of MMS22L depletion on RAD51 levels at stalled replication forks. Quantification shows average relative RAD51 abundance, n = 2; error bars, SEM.(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.D, E Effect of MMS22L depletion on RAD51 levels at stalled replication forks. Quantification shows average relative RAD51 abundance, n = 2; error bars, SEM.
Figure 4A Representative immunofluorescence images of foci induced by short CPT treatment (50 nM, 1 h) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci. Scale bar: 5 μm.B Boxplot quantification of representative experiment in (A) (n = 3; nnuclei ≥ 78). Boxes indicate the 25-75 percentile and whiskers the 10-90 percentile; horizontal lines mark  the medians. Nuclei with ≥ 25 γH2AX foci (marker of replication stress sites) were analyzed. Statistical analysis: Mann-Whitney U test; *** P ≤ 0.0001.C, D Electron micrographs of a representative reversed fork (C) and a fork with extended ssDNA region at the junction (D) (arrow). P, parental duplex; R, regressed arm.E Frequency of reversed replication forks detected by EM. Similar results were obtained in one independent experiment; n = 2.F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h. In brackets, the total number of analyzed molecules is given. Statistical analysis according to Mann-Whitney U test; ** P ≤ 0.01, n = 2.
Figure 5A Amylose pulldown of MBP (control) or MBP-tagged MMS22L-TONSL and RAD51. Eluate proteins were detected by silver staining. MT: MBP-MMS22L-GST-TONSL.B Quantitative analysis of RAD51 binding to MMS22L-TONSL such as shown in Figure EV6A. Averages shown, n = 2; error bars, SEM.C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.G Streptavidinpulldown of HSS-tagged MMS22L variants stably integrated and overexpressed in U2OS cells. Input and co-precipitated proteins were detected by immunoblotting.H Representative immunofluorescence images of CPT-induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA-resistant variants of HSS-MMS22L treated with 50 nM CPT for 1 hour. Scale bar: 5 μm.I-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.