Rab18 depletion affects stress fibers formation on L-shaped micropattern and traction force generation. (a) U2OS cells were plated onto fibronectin-coated L-shaped micropatterns and left to adhere for 3.5 h before fixation and staining with rhodamine-conjugated phalloidin. The images illustrate examples of cells on micropatterns with “straight” or “curved” hypotenuse. The curved phenotype includes both cells with the collapsed hypotenuse (collapsed) or partially spread (unspread). Scale bar: 10 µm. (b) The graph shows the percentage of collapsed or unspread cells in samples silenced for Rab18 using siRNA Rab18 or siRNA Rab18_1 (mean ± SEM). n > 80 cells from three independent experiments. (c) U2OS cells transfected with siRNA control or siRNA Rab18 were plated on fibronectin-coated micropillars and imaged over time. The white lines indicate the cell borders at T0 (dotted line) and after 12 min (solid line). Red arrows indicates the cell trajectory. Scale bar: 5 µm. (d) Quantification of the traction forces within the cells. The graph represents the mean ± SEM of n > 20 cells from three independent experiments. A pairwise t test (P < 0.001) was performed with Mathematica software. The nonparametric rank test (Wilcoxon media test) was also significant (P < 0.001).
