FIGURE 5.

Soft substrates induce overall increased transcription in hUCM-MSCs in a Cofilin-1-dependent manner. (A) Representative fluorescence microscopy images of the nuclei of cells cultured on glass coverslips or 40:1 PDMS. After 48 h in culture, hUCM-MSCs were incubated with FUrd during 15, 30, and 45 min, fixed and stained with an anti-BrdU antibody that recognises FUrd (green) to identify the new transcripts and nuclei were counterstained with DAPI (blue). (B) Linear regression of FUrd nuclear incorporation (CTCF, corrected total cell fluorescence) as a function of time occurring in cells on each substrate (data represent mean ± SEM of 6 independent experiments). Linear regression analysis (using the linear regression tools of GraphPad Prism 8) shows that the slopes of the two curves are significantly different from each other (p = 0.0173), indicating increased transcriptional activity in cells cultured on soft PDMS substrates (blue line) when compared with glass (red). (C) Representative fluorescence microscopy images of FUrd incorporation during 15 and 45 min in control and Cofilin-1 knock-down cells (using siRNA). Cells were immunostained with anti-Cofilin-1 (green) and anti-BrdU/FUrd (red) antibodies and nuclei were counterstained with DAPI (blue). (D) Bars represent the mean ± SEM of the slope values of FUrd incorporation (as determined in B) for each of the indicated conditions. Only cells effectively knocked-down for Cofilin-1 (representative images highlighted with circles) were taken into account during corrected total cell fluorescence quantification of FUrd. Statistical analysis was performed for 6 independent experiments using One-Way ANOVA followed by Dunnett’s multiple comparisons test comparing all conditions against 40:1 PDMS (ns, non-significant; *p < 0.05; ***p < 0.001).