Figure 2: Actin cytoskeleton and focal adhesion organization depends on substrate stiffness.

(a–d) REF-52 cells stained for actin filaments (F-actin) on soft (9 nN μm⁻¹—(a)) and stiff (43—(b), 64—(c) and 85nN μm⁻¹—(d)) micropillar substrates. (e–h) Corresponding orientation plots for actin staining, where the different colours indicate different orientations of actin filaments as per the given colourmap. Actin stress fibre microdomains can be identified by the uniformly coloured zones in the orientation plots. (i) Live-cell image of REF-52 cell transfected with RFP-Ftractin (green) and YFP-Paxillin (magenta), on soft substrate (9 nN μm⁻¹). (j) Focal adhesion (FA) area as function of substrate stiffness, k. Each boxplot corresponds to at least 500 FAs from five cells.• represents the mean areas. Box ends represent the first and third quartiles of the data, and whisker ends represent the last data within 1.5 interquartile range. (k) Area of actin microdomains relative to cell area, A_r, as a function of substrate stiffness, k. Each data point represents 25–30 cells and error bars represent s.e. of mean. P values were calculated using Mann–Whitney U-test. *P<0.05; ^**P<0.01; ^***P<0.0001. Scale bars, 20 μm.