Fig. 7. SMER28 enhances degradation of short-lived misfolded proteins by UPS in VCP and UFD1L/NPL4 dependent manner.

a, b HeLa cells (a) or primary cortical neurons (b) were pre-treated with 20 µM SMER28 or DMSO for 4 h, followed by treatment with puromycin for indicated time points, n = 3; (a) P = 0.0478, (b) P = 0.0022. c HeLa cells were pre-treated for 1 h with 20 µM SMER28 or DMSO in a medium deprived of methionine, followed by the addition of methionine analog L-azidohomoalanine (AHA) for indicated time points. Cells were lysed and analyzed by Western blotting using streptavidin antibody, n = 3. d, e HeLa cells were pre-treated with 20 µM SMER28, 10 µM NW1030, or DMSO for 4 h, followed by treatment with puromycin for 15 min, n = 5; quantification in (e one-way ANOVA: P < 0.0001 with post hoc Tukey test, SMER28 P < 0.0001, NW1030 P = 0.0213). f–i HeLa cells was treated with siRNA against VCP (f, g) and UFD1L or NPL4 (h, i). After 48 h cells were pre-treated with 20 µM SMER28 or DMSO for 4 h, followed by treatment with puromycin for 15 min, n = 3; quantification in (g one-sample t test, Ctrl P = 0.0442) and (I SMER28 P = 0.0175). j, k HeLa cells were pre-treated with 20 µM SMER28 or DMSO for 3 h, followed by 1 h pre-treatment with 2 µM Lactacystin and 15 min with puromycin, n = 3; quantification in (k, one sample t test, DMSO P = 0.0026). l, m ATG16L1 knockout, and control cells were pre-treated with 20 µM SMER28 or DMSO for 4 h, followed by treatment with puromycin for 15 min, n = 3; quantification in (m one sample t test, control P = 0.0415, ATG16 KO P = 0.046). (a, b, d, f, h, j, l) Cells were lysed and analyzed by Western blotting. Puromycin-labeled proteins were detected using anti-puromycin antibody. Bar graphs data presented as normalized mean ± SEM. *P < 0.05, **P < 0.001, ***P < 0.0001, paired two-tailed Student’s t test unless stated otherwise; *, unspecific band; ns not significant. Source data are provided as a Source Data file.