Figure 7: FLII is required for P-Rex1-driven cell contraction.

(a) Fibroblast-collagen matrices were treated with ethanol (−dox) or 1 μg ml⁻¹ doxycycline (+dox) to induce expression of indicated constructs and scans were taken at 0 and 24 h post treatment. (b) Quantification of cell contraction depicted in a normalized to −dox treated EV control. Graph represents the average per cent matrix contraction±s.e.m. from three independent experiments. (c) CHL1 cells were transfected with NT, or two different siRNAs against human P-Rex1 (hP-siRNA5, hP-siRNA6). Levels of endogenous pMLC and P-Rex1 were detected by western blot analysis. (d) Quantification of pMLC levels in CHL1 cells described in c normalized to α-Tubulin and pMLC levels detected in the NT control. Graph represents average pMLC levels±s.e.m. from three independent experiments. (e) Collagen picrosirius staining of matrices described in a. Scale bar, 50 μm. (f) Quantification of picrosirius staining depicted in e normalized to −dox treated EV control. Graph represents the area covered by collagen I and III±s.e.m. from three independent experiments. (g) Representative GLCM images of matrices described in a. Scale bar, 100 μm. (h,i) GLCM correlation decay curves of matrices described in a showing −dox (h) and+dox (i) curves. (j) 1° human fibroblasts were either mock transfected or treated with siRNA against human FLII (hFLII siRNA1) in the presence of dox to induce expression of indicated constructs. Levels of endogenous pMLC and FLII were detected by western blot analysis. For c,j, α-Tubulin was used as a loading control. (k) Quantification of pMLC levels in 1° human fibroblasts described in j normalized to α-Tubulin and pMLC levels detected in the EV control. Graph represents average pMLC levels±s.e.m. from three independent experiments. For b,d,f,k, Student’s t-test was used to assess significance as indicated on graphs. P values indicated above each bar are relative to −dox treated EV control (b,f), NT control (d) or mock treated EV control (k). NS, non-significant; *=P≤0.05; **=P≤0.01; ***=P≤0.001.