Figure 8
Cellular traction forces are dependent on ROCK activity and actomyosin contractility but independent of FAK activity. (A–C) Phase contrast images (top) and traction force maps (bottom) calculated for SKOV3 cells adhered to FN-coated 25 kPa hydrogels following 30 minute treatment with 25 μM blebbistatin (A), 10 μM fasudil (B), and 0.25 μM PF-271 (C). (D) Maximum traction forces were analyzed following 30 minute treatment with 0.1% DMSO or the indicated compound (μM) (n = 10, 11, or 21 cells for blebbistatin, fasudil, or PF-271, respectively; *p = 0.002; **p < 0.0001 using the Wilcoxon matched pairs test). (E) SKOV3 cells plated on 25 kPa hydrogels were treated with 0.1% DMSO (Ctrl), 25 μM blebbistatin (Bleb), 10 μM fasudil (Fas), or 0.25 μM PF-562271 (PF271) for 30 minutes before being fixed and stained to visualize vinculin (Vinc) and FAK phosphorylated on Tyr397 (pFAK; pY397). (F) Individual focal adhesions were outlined by thresholding vinculin images and running a custom macro in ImageJ which transferred each focal adhesion ROI to the raw vinculin and pFAK images and measured each signal intensity. Mean pFAK intensity was normalized to mean Vinc intensity within each adhesion. Histograms represent binned, normalized pFAK levels within adhesion for each treatment compared to DMSO control (n ≥ 800 adhesions from ≥8 cells per treatment). (G) SKOV3 cells were plated and treated as in panel (E) then stained for F-actin and phospho-MLC. (H) Linescans of relative F-actin and phospho-MLC intensity (in arbitrary units (a.u.)) for representative control and fasudil treated cells were plotted from the dotted lines shown in (G). (I) Pearson’s cross-correlation analysis of phospho-MLC and F-actin intensity linescans as shown in (H). The graph depicts all of the data points (green symbols), with box & whisker plots showing maximum & minimum values (whiskers), the 25th to 75th percentiles (box), and the median value (box line) (n = 22 linescans (1 scan/cell) from 3 experiments; p < 0.0001 using the Kruskal-Wallis test with multi-comparisons (unannotated differences are not significant)).
