Fig. 5: TGF-β signaling is regulated by TubA via acetylation of Smad3.

a–e 4-d-old C2C12 myoblasts pretreated for 24 h with either HDAC6 inhibitor (TubA, 5 μM), or selective inhibitor of TGF-β1 (SB43, 5 μM) or with DMSO (CTL). Myoblasts were then treated for 30 min with recombinant human TGF-β1 (rhTGF-β1; 10 ng/mL). a Myoblasts were double-stained with antibodies against Smad2/3 (in green) and acetylated tubulin (ac-tub, in red). Nuclei were labeled with DAPI (in blue). Scale bars: 50 µm. b Graphical summary of nuclear distribution of Smad2/3 fluorescence intensity from 3 independent experiments; two-way ANOVA (TubA+rhTGF-β1 versus DMSO + rhTGF-β1). c Levels of Smad2/3 phosphorylation (pSmad), Smad2/3 acetylation (ac-Smad), Smad2/3, acetylated α- tubulin (ac-tub), and α-tubulin (α-tub) were visualized by Western blot analysis. To evaluate levels of Smad2/3 phosphorylations (d) and Smad3 acetylation (e) in C2C12 cells quantifications have been performed (n = 5 independent experiments quantified); Mann–Whitney U test. f, g Cellular fractionation into 3 fractions was performed: cytosolic fraction (CE), nuclear extract (NE), and chromatin extract (Chrm). f Levels of Smad3 phosphorylation (S423-425), Smad2/3, acetylated α-tubulin (ac-tub-K40), GAPDH, HIRA and histone H3 (H3) were visualized by Western blot from 3 independent experiments. g Quantifications of distribution of Smad3 phosphorylation (S423-425); two-way ANOVA. Chromatin immunoprecipitations (ChIP) was performed using antibodies against Smad2/3. qPCR ampifications of immunoprecipitated promoter regions of the MAFbx (h) or MuRF1 (i) gene were used to detect the presence of these DNA fragments in immunoprecipitates (h, i, n = 3 independent experiments); Mann–Whitney U test. *, P < 0.05. (j, k, l, m, n) 11-wk-old C57BL/10ScSn-Dmdmdx/J mice treated with vehicle-DMSO (mdx-veh) or with TubA (mdx-TubA) for 30 consecutive days have been analyzed in TA muscle by Western blot analysis (j) and quantified (k, l, m, n). To evaluate levels of Smad3 acetylation (k), Smad2 acetylation (l), Smad3 phosphorylation (m) and Smad2/3 (n) in TA muscles, quantifications have been performed (n = 4 mice per group); Mann–Whitney U test. TCE was used as a loading control for all Western blots. (d, e, k, l, m, n) Whiskers min to max; the line in the middle of the box is plotted at the median. (b, g, h, i) Data are presented as mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; n.s, not significant, P > 0.05. kDa, relative molecular weight in kiloDalton.