Fig. 2.

CSE effects on hPASMC contractile machinery. (A) hPASMCs were treated for 24 h with 15 % CSE and the effects on proteins of the contractile machinery were analyzed by fluorescence microscopy. Representative images for the staining with phalloidin-Alexa FluorTM 647 (purple), and immunostaining with p-MLC (cyan) and vinculin (yellow, white arrows pointing to focal adhesion staining) are shown (n = 4), scale bar = 10 μm. Quantification of the coherency coefficient analyzed in 32 random cells from these experiments is shown. (B) hPASMCs were pretreated for 2 h with (+) or without (−) 15 μM NAC and maintained for 24 h in the presence of 15 % CSE. Afterwards, levels of myosin light chain (MLC) and its phosphorylated form (p-MLC), and levels of myosin light chain kinase (MLCK) and phosphorylated protein kinase C (p-PKCε isoform) were analyzed. Quantification of protein levels and representative images from (n = 4–14) are shown. (C) Actin dynamic was examined by means of flow cytometry in cells pretreated with (+) or without (−) 15 μM NAC for 2 h then treated with cytochalasin B for 2 h and afterwards exposed to 15 % CSE for 1 h. The percentage of F-actin re-polymerization was assessed and MFI of recovering cells was adjusted by the ratio between destabilized/non-destabilized cells (n = 4–13). Statistical comparison between groups was performed using two-sided unpaired Student's t-test (A, B lower) and ANOVA test followed by Tukey's test (B upper, C) (∗)p < 0.05, (∗∗)p < 0.01, (∗∗∗∗)p < 0.0001, (ns) non-significant.