Fig. 6: VCP activation by SMER28 enhances UPS flux.

a HeLa cells stably expressing Ub-G76V-GFP were treated with 20 µM SMER28 with or without 5 µM MG132 for 6 h, followed by FACS analysis, n = 6, paired two-tailed Student’s t test; DMSO P = 0.0006; MG132 P = 0.3509. b HeLa cells stably expressing Ub-G76V-GFP were treated with 20 µM SMER28 or its analogs for 6 h, followed by FACS analysis; unpaired two-tailed Student’s t test, n = 3; SMER28 P = 0.00002, A P = 0.03, B P = 0.5516, C P = 0.3542, D P = 0.0214, E P = 0.0337, F P = 0.0344, G P = 0.748. c HeLa cells stably expressing Ub-G76V-GFP were treated with 20 µM SMER28 with or without 10 µM NMS873 and 5 µM CB-5083 for 6 h, followed by FACS analysis, n = 4; paired two-tailed Student’s t test; DMSO P = 0.0089, NMS873 P = 0.4895, CB-5083 P = 0.0319. d, e HeLa cells stably expressing Ub-G76V-GFP were treated with siRNA against VCP (d n = 4) and siRNA against UFD1L or NPL4 (e n = 5). Raw data comparing effect of the knockdown in Supplementary Fig 5h. After 48 h cells were treated with 20 µM SMER28 or DMSO for 6 h, followed by FACS analysis; data normalized to DMSO control across all samples to allow comparison of SMER28 effect sizes within and between control and siRNA treated cells; statistical analysis for d, e one-way ANOVA: P < 0.0001 with post hoc Tukey test P < 0.0001. f Proteasome activity. HeLa cells were treated with 20 µM SMER28 or DMSO for 6 h, lysed and chymotrypsin-like, caspase-like and trypsin-like proteasome activities were measured using fluorogenic peptide substrates, n = 4; one sample t test. Data in bar graphs presented as normalized mean ± SEM. *P < 0.05, **P < 0.001, ***P < 0.0001; ns not significant. Source data are provided as a Source Data file.