Fig. 3
ILK silencing hinders normal FN-dependant RhoA/ROCK/MLC pathway stimulation and FN fibrillar assembly in HIEC. (a and b) siCNS and siILK cells were grown for 48 h on uncoated or FN-coated (FN-coated; 3 μg/cm2) plastic dishes. a Histograms represent the relative levels (membranous/total) of membrane-associated active RhoA (mb) compared to RhoA and β-actin detected in whole-cell lysates (tot). Values obtained by densitometry quantification analysis of WB bands were normalized to β1 integrin (from mb fractions) and β-actin (from cell lysate). The lower panels show representative corresponding protein immunoblots. b Histograms of the relative phosphorylation status of serine19 of the myosin light chain (pS19MLC/MLC) in cells cultured under the same conditions as described in (a). The values were obtained from densitometry quantification of pS19MLC and MLC bands detected by WB in whole cell lysates. β-actin protein levels were used as the loading control. (a and b) Results are expressed as the percentage (%) of the values obtained for the siCNS cells on uncoated dishes ± SEM (n ≥ 3). * p < 0.05, in a one-way ANOVA using Tukey’s correction for multiple group comparisons. ns: not significant. The lower panel in (a) and (b) shows representative corresponding protein immunoblots. c IF microscopy images representative of the actin cytoskeleton (TRITC-phalloidin) and human FN (detected with the HFN 7.1 antibody) staining in siCNS and siILK cells grown for 24 h on serum-pretreated coverslips with or without 20 uM of the specific inhibitor Y-27632 to inhibit ROCK-mediated cell contractility. The right panels show magnification (40x objective) of FN staining, red arrows point to examples of cell-assembled FN fibrils, while the red arrowheads point to examples of non-fibrillar FN aggregates. Scale bar in (c) left panels: 20 μm; right panels: 10 μm