Journal: bioRxiv

Article Title: CaMKII-RNMT axis directs activity-dependent control of RNA dynamics in neurons

doi: 10.1101/2025.10.30.685591

Figure Lengend Snippet: A) Immunoflourecence (IF) staining in adult mouse brain sections using polyclonal anti-mouse RNMT raised in rabbit. RNMT (Green), DAPI (blue), MAP2 (red). (B) RNMT IF using polyclonal anti-human RNMT raised in sheep and GFP-RNMT localisation in undifferentiated hiPSC, GFP (green), RNMT (red), DAPI (blue).

Article Snippet: Immunoprecipitations were carried out overnight at 4 °C using 1 μg of polyclonal anti-GFP (Bio-Rad) antibody and 10 μl of protein G Dynabeads (Thermofisher Scientific).

Techniques: Staining

Journal: bioRxiv

Article Title: CaMKII-RNMT axis directs activity-dependent control of RNA dynamics in neurons

doi: 10.1101/2025.10.30.685591

Figure Lengend Snippet: (A) Endogenous RNMT and GFP-RNMT localisation in hiPSC-derived Dopaminergic (DA) neurons. GFP (green), RNMT staining using polyclonal anti-human RNMT raised in sheep (red), DAPI (blue). B) Primary murine neurons transfected with GFP-RNMT visualised at 7 days in vitro (DIV). GFP-RNMT expression visualised directly by fluorescence microscopy. C) RNMT in Synapsin I-positive neurites and MAP2-positive dendrites in DA neurons. RNMT staining using polyclonal anti-human RNMT raised in sheep (green), MAP2 (red), Synapsin I (red), DAPI (blue).

Article Snippet: Immunoprecipitations were carried out overnight at 4 °C using 1 μg of polyclonal anti-GFP (Bio-Rad) antibody and 10 μl of protein G Dynabeads (Thermofisher Scientific).

Techniques: Derivative Assay, Staining, Transfection, In Vitro, Expressing, Fluorescence, Microscopy

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Filter trap of control polyQ19::YFP and expanded polyQ67::YFP (detected by anti-GFP antibody) expressed under neuronal-specific promoter in C. elegans . Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin loading control) to Q67 + Vector RNAi (mean ± s.e.m., n = 5 independent experiments). b , Percentage of nose touch responses/total trials per worm at days 1, 5 and 7 of adulthood ( n = 80 worms per condition; each worm was tested 10 times to determine the response percentage). The box plots represent the 25th–75th percentiles, the lines depict the median and the whiskers show the minimum–maximum values. c , Chemotaxis index toward 0.5% benzaldehyde at days 1, 5 and 7 of adulthood (mean ± s.e.m., n = 3 independent experiments; 65–206 worms were scored per condition for each independent experiment). In a – c , RNAi was initiated after development. d , Filter trap analysis of polyQ67::YFP aggregates (detected by anti-GFP antibody) in worms expressing endogenous WT or Ub-less mutant EPS-8(K524R/K583R/K621R) at days 1 and 3 of adulthood. Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated and total polyQ67 levels (corrected for α-tubulin) to day 1 adult Q67;EPS-8 (WT) worms (mean ± s.e.m., n = 4 independent experiments). e , Percentage of nose touch responses/total trials per worm at days 1 and 3 of adulthood ( n = 50 worms per condition). The box plots represent the 25th–75th percentiles, the lines depict the median and the whiskers show the minimum–maximum values. f , Chemotaxis index toward 0.5% benzaldehyde at days 1 and 3 of adulthood (mean ± s.e.m., n = 3 independent experiments; 68–204 worms were scored per condition for each independent experiment). Statistical comparisons were made by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test ( a ), two-way ANOVA with Sidakʼs multiple comparisons test ( b , c ) and two-way ANOVA with Fisher’s least significant difference (LSD) test ( d – f ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Control, SDS Page, Plasmid Preparation, Chemotaxis Assay, Expressing, Mutagenesis

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , eps-8 knockout reduces polyQ aggregation. Left: Filter trap assay with anti-GFP in day-5 adult worms. Right: SDS–PAGE with antibodies to GFP and α-tubulin loading control. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin) to Q67 (mean ± s.e.m., n = 6 independent experiments). b , Western blot analysis of day-5 adult worms expressing polyQ19::YFP or polyQ67::YFP peptides in neurons, using antibodies against GFP and α-tubulin loading control. Worms were treated with RNAi after development. Graph represents the relative percentage values of insoluble polyQ levels (corrected for α-tubulin) to Q19 + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). c , Filter trap assay with anti-GFP in day-5 adult polyQ67::YFP worms following RNAi treatment against RAC orthologs after development. Right: SDS–PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 3 independent experiments). d , Knockdown of IFB-2 after development does not reduce aggregation of polyQ-expanded peptides in the neurons of day-5 adult worms. Representative of 7 independent experiments. e , Knockdown of IFB-2 after development does not decrease polyQ-expanded aggregation in the muscle of day-5 adult worms. Representative of 3 independent experiments. f , Knockdown of IFB-2 after development does not decrease polyQ-expanded aggregation in the intestine of day-5 adult worms. Representative of 3 independent experiments. g , Filter trap of neuronal polyQ67::YFP aggregation with anti-GFP in day-5 adult worms upon neuronal knockdown of eps-8 and RAC orthologs. Right: SDS–PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 4 independent experiments). Statistical comparisons were made by two-tailed Wilcoxon signed-rank test ( a ), two-way ANOVA with Fisher’s LSD test ( b ), one-way ANOVA with Tukey’s multiple-comparison test ( c ), and one-way ANOVA with Dunnett’s multiple-comparison test ( g ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Knock-Out, TRAP Assay, SDS Page, Control, Western Blot, Expressing, Plasmid Preparation, Knockdown, Two Tailed Test, Comparison

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Filter trap of day 5 adult C. elegans expressing polyQ44::YFP in the intestine (detected by anti-GFP antibody). Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ44 and total polyQ44 levels (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 4 independent experiments). b , Filter trap of day 5 adult C. elegans expressing polyQ40::YFP in the muscle (detected by anti-GFP antibody). Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ40 and total polyQ40 levels (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 4 independent experiments). c , Body bends per second in worms expressing polyQ40 in the muscle at days 1, 5 and 7 of adulthood (day 1 (D1) + Vector RNAi: n = 41 worms; D1 + eps-8 RNAi: n = 41; D1 + mig-2 RNAi: n = 46; D1 + rac-2 RNAi: n = 44; D5 + Vector RNAi: n = 58 worms; D5 + eps-8 RNAi: n = 52; D5 + mig-2 RNAi: n = 48; D5 + rac-2 RNAi: n = 59; D7 + Vector RNAi: n = 42 worms; D7 + eps-8 RNAi: n = 49; D7 + mig-2 RNAi: n = 54; D7 + rac-2 RNAi: n = 49). The box plots represent the 25th–75th percentiles, the line depicts the median and the whiskers show the minimum–maximum values. d , Knockdown of eps-8 or RAC orthologs ameliorates aggregation of ALS-related mutant FUS P525L variant in the neurons of day 5 adult C. elegans (detected by anti-FUS antibody). Right: SDS-PAGE with antibodies to FUS and α-tubulin. Graphs represent the relative percentage values of aggregated and total FUS P525L protein levels (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 4 independent experiments). e , Knockdown of eps-8 or RAC orthologs ameliorates aggregation of ALS-related mutant TDP-43 M337V variant in the neurons of day 5 adult worms (detected by anti-TDP-43 antibody). Right: SDS-PAGE with antibodies to TDP-43 and α-tubulin. Graphs represent the relative percentage values of aggregated and total TDP-43 M337V protein levels (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 4 independent experiments). f , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing WT FUS or ALS-related mutant FUS R522G and FUS P525L variants (D1: n = 40 worms per condition; D5: n = 80; D7: n = 80). The box plots represent the 25th–75th percentiles, the line depicts the median and the whiskers show the minimum–maximum values. g , Chemotaxis index of FUS-ALS worm models toward 0.5% benzaldehyde at day 5 of adulthood (mean ± s.e.m., n = 3 independent experiments; 72–148 worms were scored per condition for each independent experiment). h , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing WT TDP-43 or ALS-related mutant TDP-43 M337V variant (D1: n = 30 worms per condition; D5: n = 70; D7: n = 70). The box plots represent the 25th–75th percentiles, the line depicts the median and the whiskers show the minimum–maximum values. i , Chemotaxis index of TDP-43 ALS worm models toward 0.5% benzaldehyde at day 5 of adulthood (mean ± s.e.m., n = 3 independent experiments; 48–439 worms were scored per condition for each independent experiment). j , Number of GABAergic neurons ( unc-25p ::GFP) in the nerve cord of TDP-43 ALS worms at day 5 of adulthood (mean ± s.e.m., TDP-43(WT) + Vector RNAi: n = 88 worms from two independent experiments; TDP-43(WT) + eps-8 RNAi: n = 50; TDP-43(WT) + mig-2 RNAi: n = 49; TDP-43(WT) + rac-2 RNAi: n = 49; TDP-43(M337V) + Vector RNAi: n = 73; TDP-43(M337V) + eps-8 RNAi: n = 45; TDP-43(M337V) + mig-2 RNAi: n = 53; TDP-43(M337V) + rac-2 RNAi: n = 49). The box plots represent the 25th–75th percentiles, the line depicts the median and the whiskers show the minimum–maximum values. k , Graph represents the percentage of worms displaying discontinuities in the nerve cord at day 5 of adulthood (percentage from 49–88 worms per condition from two independent experiments). Statistical comparisons were made by one-way ANOVA with Dunnett’s multiple comparisons test ( a , b , d , e ), two-way ANOVA with Sidakʼs multiple comparisons test ( c , f – j ) and two-sided Fisher’s exact test from contingency table analysis of number of worms displaying discontinuities in the nerve cord ( k ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Expressing, SDS Page, Plasmid Preparation, Knockdown, Mutagenesis, Variant Assay, Transgenic Assay, Chemotaxis Assay

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Filter trap analysis of muscle polyQ40::YFP aggregates (detected by anti-GFP antibody) in worms expressing endogenous wild-type (WT) or Ub-less mutant EPS-8(K524R/K583R/K621R) at day 1 and 3 of adulthood. Right: SDS–PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated and total polyQ40 levels (corrected for α-tubulin) to day-1 adult Q40;EPS-8 (WT) worms (mean ± s.e.m., n = 3 independent experiments). b , Body bends per second in wild-type EPS-8 and Ub-less EPS-8 worms expressing polyQ40 in muscle cells. Day 1 (D1) EPS-8(WT): n = 46 worms; D1 EPS-8(Ub-less): n = 41; D3 EPS-8(WT): n = 51; D3 EPS-8(Ub-less): n = 45. c , Body bends per second in wild-type worms and transgenic worms expressing wild-type TDP-43 (WT) or ALS-related mutant TDP-43 M337V variant. D1 wild-type + Vector RNAi: n = 44 worms; D1 wild-type + eps-8 RNAi: n = 47; D1 wild-type + mig-2 RNAi: n = 41; D1 wild-type + rac-2 RNAi: n = 42; D1 TDP-43(WT) + Vector RNAi: n = 45; D1 TDP-43(WT) + eps-8 RNAi: n = 41; D1 TDP-43(WT) + mig-2 RNAi: n = 47; D1 TDP-43(WT) + rac-2 RNAi: n = 44; D1 TDP-43(M337V) + Vector RNAi: n = 45; D1 TDP-43(M337V) + eps-8 RNAi: n = 44; D1 TDP-43(M337V) + mig-2 RNAi: n = 43; D1 TDP-43(M337V) + rac-2 RNAi: n = 41; D5 wild-type + Vector RNAi: n = 42 worms; D5 wild-type + eps-8 RNAi: n = 35; D5 wild-type + mig-2 RNAi: n = 38; D5 wild-type + rac-2 RNAi: n = 36; D5 TDP-43(WT) + Vector RNAi: n = 38; D5 TDP-43(WT) + eps-8 RNAi: n = 39; D5 TDP-43(WT) + mig-2 RNAi: n = 34; D5 TDP-43(WT) + rac-2 RNAi: n = 38; D5 TDP-43(M337V) + Vector RNAi: n = 34; D5 TDP-43(M337V) + eps-8 RNAi: n = 39; D5 TDP-43(M337V) + mig-2 RNAi: n = 35; D5 TDP-43(M337V) + rac-2 RNAi: n = 38. In b,c , the box plots represent the 25th–75th percentiles, the line depicts the median and the whiskers show the minimum–maximum values. d , Chemotaxis index of FUS ALS worm models toward 0.5% benzaldehyde at day 1 of adulthood (mean ± s.e.m., n = 3 independent experiments, 66-174 worms were scored per condition for each independent experiment). No significant differences were observed. e , Chemotaxis index of TDP-43 ALS worm models toward 0.5% benzaldehyde at day 1 of adulthood (mean ± s.e.m., n = 3 independent experiments, 48-257 worms were scored per condition for each independent experiment). No significant differences were observed. In all the experiments, RNAi was initiated after development. Statistical comparisons were by two-way ANOVA with Fisher’s LSD test ( a,b ) and two-way ANOVA with Šidák multiple-comparison test ( c,d,e ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Expressing, Mutagenesis, SDS Page, Transgenic Assay, Variant Assay, Plasmid Preparation, Chemotaxis Assay, Comparison

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: 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-43 A382T . 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-43 A382T protein levels (corrected for β-actin) to NT shRNA TDP-43 A382T 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 FUS P525L . Right: SDS-PAGE with antibodies to FUS, EPS8 and β-actin loading control. Graph represents the relative percentage values of aggregated and total FUS P525L protein levels (corrected for β-actin) to NT shRNA FUS P525L 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-43 A382T (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-43 A382T protein levels (corrected for β-actin) to TDP-43 A382T 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 ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Expressing, shRNA, Construct, SDS Page, Control, Mutagenesis, Immunocytochemistry, Derivative Assay, Western Blot, Plasmid Preparation, Over Expression, Two Tailed Test

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Chymotrypsin-like proteasome activity in polyQ67::YFP C. elegans (relative slope to Vector RNAi, mean ± s.e.m., n = 12 biological replicates). b , Western blot of polyQ67::YFP C. elegans with anti-LGG-1/LC3 antibody. LC3-I is conjugated to phosphatidylethanolamine to form LC3-II, whose levels reflect the number of autophagosomes and autophagy-related structures. α-tubulin is the loading control. Graph represents the relative percentage values of LC3-II (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 6 independent experiments). In a-b , RNAi was initiated after development and worms were analyzed at day 5 of adulthood. c , Chymotrypsin-like proteasome activity in HEK293 cells (relative slope to non-targeting (NT) shRNA, mean ± s.e.m., n = 12 biological replicates). d , Chymotrypsin-like proteasome activity in HEK293 cells expressing Q100-HTT-GFP (relative slope to NT shRNA, mean ± s.e.m., n = 11 biological replicates). e , Western blot of wild-type HEK293 cells with antibodies against LC3 and β-actin loading control. Graph represents the relative percentage values of LC3-II (corrected for β-actin) to NT shRNA (mean ± s.e.m., n = 3 independent experiments). f , Western blot of HEK293 cells expressing Q100-HTT-GFP with anti-LC3 antibody. Graph represents the relative percentage values of LC3-II (corrected for β-actin) to NT shRNA (mean ± s.e.m., n = 3 independent experiments). Statistical comparisons were made by two-tailed Student’s t -test for unpaired samples ( a ), two-tailed Wilcoxon signed-rank test ( b ), and one-way ANOVA with Dunnett’s multiple-comparison test ( c-f ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Activity Assay, Plasmid Preparation, Western Blot, Control, shRNA, Expressing, Two Tailed Test, Comparison

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Filter trap of polyQ67::YFP aggregates (detected by anti-GFP antibody) in day 5 adult worms treated with 10 µM CytoD or DMSO vehicle control for 6 hours before lysis. Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin loading control) to Q67 + Vector RNAi + DMSO (mean ± s.e.m., n = 3 independent experiments). b , Filter trap with anti-GFP antibody of HEK293 human cells expressing Q23-HTT-GFP or Q100-HTT-GFP treated with 2 µM CytoD or DMSO vehicle control for 4 hours before lysis. Right: SDS-PAGE with antibodies to HTT and β-actin loading control. Graphs represent the relative percentage of aggregated and total HTT-GFP levels (corrected for β-actin) to Q23-HTT-GFP + DMSO (mean ± s.e.m., n = 3 independent experiments). c , Filter trap of mutant FUS P525L aggregates (detected by anti-FUS antibody) in day 5 adult worms treated with 10 µM CytoD for 6 hours. Right: SDS-PAGE with antibodies to FUS and α-tubulin. Graphs represent the relative percentage values of aggregated and total FUS levels (corrected for α-tubulin loading control) to FUS P525L + DMSO (mean ± s.e.m., n = 6 independent experiments). d , Filter trap of mutant TDP-43 M337V aggregates (detected by anti-TDP-43 antibody) in day 5 adult worms treated with 10 µM CytoD for 6 hours. Right: SDS-PAGE with antibodies to TDP-43 and α-tubulin. Graphs represent the relative percentage values of aggregated and total TDP-43 (corrected for α-tubulin) levels to TDP-43 M337V + DMSO (mean ± s.e.m., n = 6 independent experiments). e , Filter trap analysis of polyQ67::YFP (detected by anti-GFP antibody) in day 3 adult worms expressing endogenous WT EPS-8 or Ub-less mutant EPS-8 treated with 10 µM CytoD (6 hours). Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin) to Q67;EPS-8(WT) + DMSO (mean ± s.e.m., n = 3 independent experiments). Statistical comparisons were made by two-way ANOVA with Sidakʼs multiple comparisons test ( a ), two-way ANOVA with Fisher’s LSD test ( b , e ) and two-tailed Wilcoxon signed-rank test ( c , d ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Control, Lysis, SDS Page, Plasmid Preparation, Expressing, Mutagenesis, Two Tailed Test

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: 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.

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Plasmid Preparation, Control, Lysis, SDS Page

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Knockdown of kgb-1 after development prevents polyQ67::YFP aggregation (detected by anti-GFP antibody) in the neurons of day 5 adult C. elegans . Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated and total polyQ67 (corrected for α-tubulin loading control) to Vector RNAi (mean ± s.e.m., n = 7 independent experiments). b , Filter trap analysis of polyQ67::YFP aggregates (detected by anti-GFP antibody) in day 3 adult worms expressing endogenous WT or Ub-less mutant EPS-8 on kgb-1 RNAi treatment. Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin loading control) to Q67;EPS-8(WT) + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). c , Knockdown of kgb-1 after development ameliorates aggregation of ALS-related mutant FUS variants in the neurons of day 5 adult worms (detected by anti-FUS antibody). Right: SDS-PAGE with antibodies to FUS and α-tubulin. Graphs represent the relative percentage values of aggregated and total FUS levels (corrected for α-tubulin loading control) to WT FUS + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). d , Knockdown of kgb-1 after development decreases TDP-43 M337V aggregation in the neurons of day 5 adult worms (detected by anti-TDP-43 antibody). Right: SDS-PAGE with antibodies to TDP-43 and α-tubulin. Graphs represent the relative percentage values of aggregated TDP-43 and total TDP-43 levels (corrected for α-tubulin loading control) to WT TDP-43 + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). Statistical comparisons were made by two-tailed Wilcoxon signed-rank test ( a ), two-way ANOVA with Fisher’s LSD test ( b , d ) and two-way ANOVA with Sidakʼs multiple comparisons test ( c ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Knockdown, SDS Page, Control, Plasmid Preparation, Expressing, Mutagenesis, Two Tailed Test

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Knockdown of jnk-1 after development prevents polyQ67::YFP aggregation (detected by anti-GFP antibody) in the neurons of day-5 adult worms. Right: SDS–PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated and total polyQ67 (corrected for α-tubulin loading control) to Vector RNAi (mean ± s.e.m., n = 6 independent experiments). b , Filter trap analysis of polyQ67::YFP (detected by anti-GFP antibody) in day-3 adult worms expressing either endogenous wild-type (WT) EPS-8 or Ub-less mutant EPS-8 upon jnk-1 RNAi. Right: SDS–PAGE with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin loading control) to Q67;EPS-8(WT) + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). c , Knockdown of jnk-1 reduces aggregation of the severe FUS P525L mutant variant, but does not significantly affect aggregation of wild-type (WT) FUS or mutant FUS R522G (detected by anti-FUS antibody). Right: SDS-PAGE with antibodies to FUS and α-tubulin. Graphs represent the relative percentage values of aggregated and total FUS levels (corrected for α-tubulin loading control) to FUS WT + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). d , Knockdown of jnk-1 after development decreases TDP-43 M337V aggregation in the neurons of day-5 adult worms (detected by anti-TDP-43 antibody). Right: SDS-PAGE with antibodies to TDP-43 and α-tubulin. Graphs represent the relative percentage values of aggregated TDP-43 and total TDP-43 levels (corrected for α-tubulin loading control) to wild-type (WT) TDP-43 + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). Statistical comparisons were made by two-tailed Wilcoxon signed-rank test ( a ), two-way ANOVA with Fisher’s LSD test ( b, d ), and two-way ANOVA with Šidák multiple-comparison test ( c ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Knockdown, SDS Page, Control, Plasmid Preparation, Expressing, Mutagenesis, Variant Assay, Two Tailed Test, Comparison

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Inhibition of elevated DUB activity in day 5 adult worms ameliorates aggregation of neuronal polyQ67::YFP (detected by anti-GFP antibody). Right: SDS-PAGE with antibodies to GFP and α-tubulin loading control. Graphs represent the relative percentage values of aggregated and total polyQ67 levels (corrected for α-tubulin) to Q67 + DMSO vehicle control (mean ± s.e.m., n = 7 independent experiments). b , Inhibition of elevated DUB activity decreases aggregation of mutant FUS P525L in C. elegans neurons (detected by anti-FUS antibody). Right: SDS-PAGE of total FUS protein levels with anti-FUS antibody. Graphs represent the relative percentage values of aggregated and total FUS protein levels (corrected for α-tubulin) to WT FUS + DMSO vehicle control (mean ± s.e.m., n = 3 independent experiments). c , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing polyQ19 or polyQ67 in neurons (day 5 (D5): n = 80 worms per condition (except WT + DUB inhibitor, n = 79); D7: n = 40 worms per condition). d , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing WT FUS or ALS-related mutant FUS R522G and FUS P525L variants ( n = 40 worms per condition). e , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing WT TDP-43 or ALS-related mutant TDP-43 M337V variant ( n = 40 worms per condition). In c – e , the box plots represent the 25th–75th percentiles, the lines depict the median and the whiskers show the minimum–maximum values. Worms were treated with 13.7 µg ml −1 PR-619 (broad-spectrum DUB inhibitor) or vehicle control (DMSO) for 4 hours on day 5 of adulthood ( a ) or for 24 hours on day 4 of adulthood ( b – e ) and analyzed at the indicated ages. f , Single knockdown after development of csn-6 (mean ± s.e.m.: 20.56 days ± 0.62, P < 0.0001), F07A11.4 (mean ± s.e.m.: 22.16 ± 0.71, P < 0.0001) and usp-4 (mean ± s.e.m.: 20.42 ± 0.59, P < 0.0001) extends lifespan in WT worms compared with Vector RNAi controls (mean ± s.e.m.: 16.22 ± 0.44). By contrast, knockdown of usp-5 (mean ± s.e.m.: 16.66 ± 0.45, P = 0.4589), otub-3 (mean ± s.e.m.: 16.59 ± 0.46, P = 0.6532), math-33 (mean ± s.e.m.: 15.31 ± 0.36, P = 0.0552) or usp-48 (mean ± s.e.m.: 16.67 ± 0.36, P = 0.8363) does not affect lifespan. P values: two-sided log-rank test, n = 96 worms per condition. Supplementary Table contains statistical analysis and replicate data from independent lifespan experiments. g , Knockdown of usp-4 after development prevents polyQ67::YFP aggregation in day 5 adult worms (detected by anti-GFP antibody). Right: SDS-PAGE with antibodies to GFP and α-tubulin. Graph represents the relative percentage values of aggregated and total polyQ67 protein levels (corrected for α-tubulin) to Q67 + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). Statistical comparisons were made by two-tailed Wilcoxon signed-rank test ( a ), two-way ANOVA with Fisher’s LSD test ( b ), two-way ANOVA with Sidakʼs multiple comparisons test ( c – e ), two-sided log-rank test ( f ) and one-way ANOVA with Dunnett’s multiple comparisons test ( g ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Inhibition, Activity Assay, SDS Page, Control, Mutagenesis, Transgenic Assay, Expressing, Variant Assay, Knockdown, Plasmid Preparation, Two Tailed Test

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: a , Knockdown of usp-4 ameliorates mutant FUS aggregation in the neurons of day 5 adult C. elegans (detected by anti-FUS antibody). Right: SDS-PAGE with antibodies to FUS and α-tubulin. Graphs represent the relative percentage of aggregated and total FUS levels (corrected for α-tubulin) to WT FUS + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). b , Knockdown of usp-4 decreases mutant TDP-43 M337V aggregation in day 5 adult worms (detected by anti-TDP-43 antibody). Right: SDS-PAGE with antibodies to TDP-43 and α-tubulin. Graphs represent the relative percentage of aggregated and total TDP-43 levels (corrected for α-tubulin) to TDP-43(WT) + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). c , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing polyQ19 or polyQ67 in neurons (day 1 (D1): n = 40 worms per condition; D5: n = 80; D7: n = 80). d , Chemotaxis index of neuronal polyQ-expressing worms toward 0.5% benzaldehyde (mean ± s.e.m., n = 3 independent experiments; 56–215 worms were scored per condition for each independent experiment). e , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing WT FUS or ALS-related mutant FUS R522G and FUS P525L variants (D1: n = 40 worms per condition; D5: n = 80; D7: n = 80). f , Chemotaxis index of FUS-ALS worm models toward 0.5% benzaldehyde (mean ± s.e.m., n = 3 independent experiments; 78–150 worms were scored per condition for each independent experiment). g , Percentage of nose touch responses/total trials in WT worms and transgenic worms expressing WT TDP-43 or ALS-related mutant TDP-43 M337V variant (D1: n = 30 worms per condition; D5: n = 70; D7: n = 70). In c , e , g , the box plots represent the 25th–75th percentiles, the lines depict the median and the whiskers show the minimum–maximum values. h , Western blot with antibody to EPS-8 of day 10 adult worms on usp-4 knockdown. RNAi was initiated after development. Graph: relative percentage values of EPS-8 protein levels (corrected for α-tubulin) to Vector RNAi (mean ± s.e.m., n = 6 independent experiments). i , Knockdown of usp-4 after development prolongs lifespan in worms expressing WT EPS-8 ( P < 0.01) but not the short lifespan of Ub-less EPS-8 mutants ( P = 0.6798). EPS-8(WT) + Vector RNAi: 21.01 days ± 0.46, EPS-8(WT) + usp-4 RNAi: 23.27 ± 0.40, EPS-8(Ub-less) + Vector RNAi: 17.51 ± 0.55, EPS-8(Ub-less) + usp-4 RNAi: 18.24 ± 0.48. P values: two-sided log-rank test, n = 96 worms per condition. Supplementary Table contains statistical analysis and replicate data of independent lifespan experiments. j , Knockdown of usp-4 prevents polyQ67::YFP aggregation (detected by anti-GFP antibody) in worms expressing WT EPS-8 but not in worms expressing Ub-less EPS-8 mutant. Right: western blot with antibodies to GFP and α-tubulin. Graphs represent the relative percentage values of aggregated polyQ67 and total polyQ67 levels (corrected for α-tubulin loading control) to Q67;EPS-8(WT) + Vector RNAi (mean ± s.e.m., n = 3 independent experiments). In all the experiments, RNAi was initiated after development. Statistical comparisons were made by two-way ANOVA with Sidakʼs multiple comparisons test ( a , c , e – g ), two-way ANOVA with Fisher’s LSD test ( b , d , j ), two-tailed Wilcoxon signed-rank test ( h ) and two-sided log-rank test ( i ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Knockdown, Mutagenesis, SDS Page, Plasmid Preparation, Transgenic Assay, Expressing, Chemotaxis Assay, Variant Assay, Western Blot, Control, Two Tailed Test

Journal: Nature Aging

Article Title: The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation

doi: 10.1038/s43587-025-00943-w

Figure Lengend Snippet: 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 FUS P525L 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-43 A382T 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 ).

Article Snippet: For both filter trap and western blot analyses of C. elegans , immunoblotting was performed with antibodies against GFP (AMSBIO, TP401, dilution 1:5,000), FUS (Abcam, ab154141, clone CL0190, 1:1,000) and TDP-43 (Abcam, ab225710, 1:1,000).

Techniques: Western Blot, Expressing, Control, shRNA, Lysis, Co-Immunoprecipitation Assay, Knockdown, SDS Page, Over Expression, Mutagenesis, Immunocytochemistry, Derivative Assay