Fig. 4

Immunofluorescence images showing the distribution of MRTFA in the cells. a Cells are actuated for 0, 4, 8, 12 and 22 h (340 mW, 1 Hz, 100 ms laser ON time), b The actuation amplitude is varied by using a laser power of 100 mW, 260 mW or 340 mW (1 Hz, 100 ms ON time, 12 h actuation), or by reducing the laser pulse to 20 ms at 340 mW laser power and 1 Hz frequency. c Cells are actuated at 0.1 Hz and 0.5 Hz at 340 mW (100 ms ON time,12 h actuation), while higher frequencies of 5 Hz and 10 Hz are obtained using a shorter ON time of 20 ms, keeping the laser power constant (340 mW). d After actuating for 12 h (340 mW, 1 Hz, 100 ms laser ON time), actuation is ceased and cells are allowed to relax for 2.5, 5, 10 and 14 h. e–j The nuclear MRTFA % is measured to assess the effect of e duration of actuation, f actuation amplitude by changing laser output, g actuation amplitude by changing the ON time of the laser pulse, h frequency when ON time = 100 ms, i frequency when ON time = 20 ms and j relaxation. Scale bar = 20 µm. The box plots show the nuclear MRTFA % for each cell (n ≥ 3, N ≥ 60 cells, except e, where N ≥ 48 cells). In the box plots, the interquartile range (IQR) between the first and the third quartiles is indicated by the box, while whiskers denote 1.5 IQR. The hollow square, the horizontal line, and the filled dots represent the average, the median, and the outliers, respectively. On the left of the box plot, all data points are shown, the normal distribution curve serves to guide the eye. *, **, *** are determined using one way ANOVA or Welch test, depending on the homogeneity of variances, and represent statistical significance at p < 0.05, 0.01 and 0.001, respectively