Fig 2: USP4 knockdown decreases EPS8 levels and disease-related protein aggregation in human cells.a, Western blot analysis of EPS8 levels in HEK293 cells expressing control NT or USP4 shRNA. Cells were treated with 0.5 µM MG-132 proteasome inhibitor or DMSO vehicle control for 16 hours before the lysis. β-Actin is the loading control. Representative of three independent experiments. b, Co-IP with control IgG and antibody against USP4 in HEK293 cells. Co-IP was followed by western blot with antibodies to USP4 and EPS8. Representative of two independent experiments. c, Filter trap with anti-GFP of HEK293 cells expressing either control Q23-HTT-GFP or aggregation-prone Q100-HTT-GFP upon knockdown of USP4 using two independent shRNAs. Right: SDS-PAGE with antibodies to HTT, EPS8, USP4 and β-actin loading control. Graphs: mean ± s.e.m. relative percentage of aggregated Q100-HTT and total Q100-HTT or EPS8 levels (corrected for β-actin) to NT shRNA Q100-HTT cells (mean ± s.e.m., n = 3 independent experiments). d, Filter trap of Q100-HTT-GFP aggregation (detected with anti-GFP antibody) in HEK293 cells upon knockdown of USP4 and overexpression of EPS8. Right: SDS-PAGE with antibodies to HTT, EPS8, USP4 and β-actin loading control. Graphs: mean ± s.e.m. relative percentage of aggregated and total Q100-HTT levels (corrected for β-actin) to NT shRNA Q100-HTT cells (mean ± s.e.m., n = 3 independent experiments). e, Filter trap with anti-FUS of HEK293 cells expressing aggregation-prone FUSP525L upon knockdown of USP4. Right: SDS-PAGE with antibodies to FUS, EPS8, USP4 and β-actin loading control. Graphs: mean ± s.e.m. relative percentage of aggregated FUS and total FUS or EPS8 levels (corrected for β-actin) to NT shRNA cells (mean ± s.e.m., n = 4 independent experiments). f, Filter trap with anti-TDP-43 antibody of HEK293 cells expressing aggregation-prone mutant TDP-43A382T upon knockdown of USP4. Right: SDS-PAGE with antibodies to TDP-43, EPS8, USP4 and β-actin loading control. Graphs: mean ± s.e.m. relative percentage of aggregated TDP-43 and total TDP-43 or EPS8 levels (corrected for β-actin) to NT shRNA cells (mean ± s.e.m., n = 3 independent experiments). g, Immunocytochemistry of FUS(P525L) ALS iPSC-derived motor neurons with anti-cleaved caspase-3 (red), anti-MAP2 (green) and Hoechst (nucleus, blue). Scale bar, 20 µm. Graph represents the percentage of cleaved caspase-3-positive cells/total nuclei (mean ± s.e.m. of four biological replicates from two independent experiments, NT shRNA: 185 total nuclei; USP4 shRNA 1: 149 total nuclei; USP4 shRNA 2: 147 total nuclei). Statistical comparisons were made by one-way ANOVA with Dunnett’s multiple comparisons test (c,e–g) and two-way ANOVA with Tukeyʼs multiple comparisons test (d).Source data

Fig 3: Cytochalasin D treatment reduces polyQ100-HTT aggregation induced by EPS8 overexpression in human cells.Filter trap with anti-GFP antibody of Empty Vector or EPS8 overexpressing (OE) Q100-HTT-GFP HEK293. Cells were treated with 2 µM CytoD or DMSO vehicle control for 4 h before lysis. The graph represents the relative percentage of aggregated Q100-HTT levels to Empty Vector + DMSO (mean ± s.e.m., n = 3 independent experiments). Statistical comparisons were made by two-way ANOVA with Fisher’s LSD test. Right: SDS-PAGE with antibodies to EPS8 and β-actin loading control. Source data

Fig 4: EPS8/RAC signaling modulates disease-related protein aggregation in human cells.a, Filter trap with anti-GFP antibody of HEK293 human cells expressing Q23-HTT-GFP or Q100-HTT-GFP treated with either non-targeting (NT) shRNA or independent shRNA constructs against EPS8. Right: SDS-PAGE with antibodies to HTT, EPS8 and β-actin loading control. Graphs represent the relative percentage values of aggregated and total Q100-HTT protein levels (corrected for β-actin) to NT shRNA Q100-HTT cells (mean ± s.e.m., n = 5 independent experiments). b, Filter trap with anti-TDP-43 antibody of HEK293 cells expressing WT TDP-43 or ALS-related mutant TDP-43A382T. Right: SDS-PAGE with antibodies to TDP-43, EPS8 and β-actin loading control. Graphs represent the relative percentage values of aggregated and total TDP-43A382T protein levels (corrected for β-actin) to NT shRNA TDP-43A382T cells (mean ± s.e.m., n = 4 independent experiments). c, Filter trap with anti-FUS antibody of HEK293 cells expressing WT FUS or ALS-related mutant FUSP525L. Right: SDS-PAGE with antibodies to FUS, EPS8 and β-actin loading control. Graph represents the relative percentage values of aggregated and total FUSP525L protein levels (corrected for β-actin) to NT shRNA FUSP525L cells (mean ± s.e.m., n = 4 independent experiments). d, Immunocytochemistry of FUS(P525L) ALS iPSC-derived motor neurons with anti-cleaved caspase-3 (red), anti-MAP2 (green) and Hoechst (nucleus, blue). Scale bar, 10 µm. Graph represents the percentage of cleaved caspase-3-positive cells/total nuclei (mean ± s.e.m. of nine biological replicates from two independent experiments, NT shRNA: 383 total nuclei and EPS8 shRNA 2: 192 total nuclei). e, Western blot analysis of FUS(P525L) ALS iPSC motor neurons with antibodies to phosphorylated RIP (P-RIP) at Ser166, total RIP and β-actin loading control. Graph represents the relative percentage ratio of P-RIP/total RIP levels to NT shRNA (mean ± s.e.m., n = 3 independent experiments). f, Increased aggregation of Q100-HTT-GFP (detected by anti-GFP antibody) in HEK293 cells overexpressing (OE) EPS8. Right: SDS-PAGE with antibodies to HTT, EPS8 and β-actin. Graphs represent the relative percentage values of aggregated and total Q100-HTT (corrected for β-actin) levels to Q100-HTT cells + empty vector (mean ± s.e.m., n = 6 independent experiments). g, Overexpression of EPS8 increases aggregation of mutant TDP-43A382T (detected by anti-TDP-43 antibody) in HEK293 cells. Right: SDS-PAGE with antibodies to TDP-43, EPS8 and β-actin. Graphs represent the relative percentage values of aggregated and total TDP-43A382T protein levels (corrected for β-actin) to TDP-43A382T cells + empty vector (mean ± s.e.m., n = 6 independent experiments). h, Filter trap with anti-GFP antibody of HEK293 human cells expressing control Q23-HTT-GFP or aggregation-prone Q100-HTT-GFP. The treatment with 2 U ml−1 RAC activator (6 hours) hastens aggregation of Q100-HTT-GFP. Right: SDS-PAGE with antibodies to HTT and β-actin. Graphs represent the relative percentage of aggregated and total HTT-GFP levels (corrected for β-actin) to Q23-HTT-GFP (PBS vehicle control) cells (mean ± s.e.m., n = 4 independent experiments). Statistical comparisons were made by one-way ANOVA with Dunnett’s multiple comparisons test (a–c,e), two-sided t-test for unpaired samples (d), two-tailed Wilcoxon signed-rank test (f,g) and two-way ANOVA with Fisher’s LSD test (h).Source data