Figure 3
Substrate rigidity enhances actin-myosin co-localization and myosin phosphorylation. (A) SKOV-3 cells plated on FN-coated glass (G), 25 kPa, or 3 kPa hydrogels were fixed and stained to visualize F-actin and dually-phosphorylated myosin light chain (ppMLC). Images of a representative cell on 25 kPa hydrogel are shown (n.b. this is the same cell shown in Fig. 2). Co-localization of F-actin and ppMLC was assessed by pixel-by-pixel image correlation analysis performed using intensity correlation analysis to produce Mander’s correlation coefficients. Average Mander’s coefficients are plotted as mean ± SEM (n= 15 cells; *p = 0.0015 using Wilcoxon test; **p < 0.0001 using Wilcoxon test; #, no significant difference between Manders’ coefficients for F-actin and ppMLC on 3 kPa gels, while the coefficients for both F-actin and ppMLC on 3 kPa are significantly different (p < 0.0001, unpaired t-test) than the corresponding values on 25 kPa and glass). (B) Lysates from SKOV-3 cells plated on the indicated substrates (as in panel (A)) were separated by SDS-PAGE and probed with antibodies against ppMLC (Thr18/Ser19) and total myosin light chain (MLC). The positions of the 20 kDa and 15 kDa molecular weight markers are indicated. (C) The relative ratio of ppMLC to total MLC was determined by densitometry (mean ± s.d.; n=6; *p = 0.005; **p = 0.006, using a Mann-Whitney test).
