Figure 3.

Soft substrates favor euchromatin and expression of endogenous pluripotency-related genes by MSCs. To assess (A) chromatin condensation and (B) euchromatin content, mean fluorescence intensity values of DAPI and H4K16ac stained nuclei (respectively) were quantified (20 nuclei per independent experiment) and represented as mean ± SEM (n = 3) in MSCs after 24 hours or 4 days in culture. Stiff substrates correspond to TCPs (A) or glass coverslips (B). (A,B) Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test when comparing cells on distinct substrates after 24 h or 4d in culture (*p < 0.01, ****p < 0.0001). (C) Representative western blot analysis of nuclear extracts obtained from MSCs cultured for 24 hours on TCPs (stiff) or PDMS (1.5 kPa, 15 kPa) probed with antibodies specific for H4K16ac and total H4, and respective quantification in (D) as fold change of H4K16ac/total H4 ratio (relative to the stiff substrate). Data represent mean ± SEM (n = 4). (E) Scatter plot relating DAPI and H4K16ac MFI (data from A and B). (F) Gene expression analysis of pluripotency-related genes (Nanog, Oct4 and Sox2) expressed by MSCs cultured for 4 days on distinct substrates was assessed by qRT-PCR. Bars represent mean ± SEM of at least 3 independent experiments and are expressed as fold change of 2^−ΔΔCt using TCPs (stiff) as the control condition. Statistical analysis was performed using Kruskal-Wallis test followed by Dunn’s multiple comparison test (*p < 0.05) in (D,F).