Figure 4
From: Adult fibroblasts use aggresomes only in distinct cell-states
p38α MAPK and p38β MAPK inhibition with TAK-715, and TAK1 inhibition with 5Z-7 suppress aggresome formation in fibroblasts. (A,B) Average DESeq2 log2(fold change) values for all MAPK cascade or Regulation of stress-activated MAPK cascade genes as proliferating (purple), senescent (blue), quiescent (red), and immortalized (orange) fibroblasts respond to MG132 treatment. (C) A schematic summarizing the interaction of TAK1, MKK3/6 and p38 MAPKs in stress-activated MAPK signaling. Inhibitors are shown in red. (D–G) Quiescent and immortalized fibroblasts were treated with 5 µg/mL 5Z-7 or 0.05% DMSO for 24 h, and then additionally treated with 10 µM MG132 for 7 h still in the presence of 5Z-7 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (N = 3; Student’s t-test; mean ± SD). (H–K) Quiescent and immortalized fibroblasts were treated with 10 µM MG132 and/or 5 µg/mL 5Z-7-Oxozeaenol for 8 h as above and then analyzed for expression of p38 MAPKs, actin, and P-p38 MAPKs (T180/Y182) by western blot. (N = 3; two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (L–O) Quiescent and immortalized fibroblasts were treated with 50 µM TAK-715 or 1% DMSO for 24 h, then additionally treated with 10 µM MG132 for 7 h still in the presence of TAK-715 or DMSO prior to being immunostained for K48 polyUb (green) and Hoechst (nucleus; blue). Samples were analyzed for the proportion of cells forming the aggresome (N = 3; Student’s t-test; mean ± SD). (P,Q) Proliferating, senescent, quiescent, and immortalized fibroblasts were treated with 0.1% DMSO or 10 µM MG132 for 8 h prior to analysis of p38 MAPKs, and P-p38 MAPKs (T180/Y182) by western blot. A ratio of P-p38/total p38 was used to measure activation of p38 (N = 3; Two-way ANOVA with post-hoc Tukey’s test; mean ± SD). (R) A schematic depicting a working model for p38 MAPK’s role in aggresome formation in primary mouse dermal fibroblasts, suggesting p38 activity is necessary but not sufficient. Scale bars 10 µm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
