FIGURE 6.

RNA polymerase II transcription elongation is faster in cells cultured on soft PDMS substrates. (A) Representative confocal microscopy images of MRC-5 cells (expressing GFP-RNA polymerase II) during fluorescence loss in photobleaching (FLIP) experiments. MRC-5 cells were cultured on stiff μ-slide glass wells or soft 1.5 kPa PDMS and after 72 h in culture cells were subjected to FLIP assay. In the zoomed images on the right panel (from the highlighted regions indicated with the yellow rectangles on the left panel), the dashed white lines circumscribe the bleached areas and the solid yellow lines represent the fluorescence measurement areas. (B) Representative three-phase exponential decay curves of GFP-RNA polymerase II signal of two single cells cultured on each substrate (glass or PDMS). (C) RNA polymerase II elongation half-life time was calculated based on the half-time slow parameter of the three-phase exponential decay curves obtained from cells cultured on stiff or soft substrates. Bars represent the median with range of at least four independent experiments. For each experiment, at least 10 cells were quantified. Statistical analysis was performed using the non-parametric Mann–Whitney test, with significant differences indicated as *p < 0.05.