Fig. 3.

Effects of nAChR inhibition on CSE-mediated alterations on hPASMC. (A) hPASMC were pre-treated with 100 nM α-bungarotoxin (α-bt) or 10 μM mecamylamine (Me) and then exposed to 15 % CSE for 48 h. X-gal staining to quantify β-galactosidase activity was analyzed by flow cytometry. Cell senescence is presented as median fold change in β-Gal MFI and normalized to the levels in non-CSE treated (−); (B) Quantification of F-actin re-polymerization percentage measured by flow cytometry. The re-polymerization analysis was assessed in cells exposed to 15 % CSE in the absence (−) or presence (+) of 100 nM α-bt or 10 μM Me. The MFI of recovering cells was adjusted by the ratio between destabilized/non-destabilized cells. Data are presented as mean ± s.e.m (n = 6–7). (C) Analysis of myosin light chain kinase (MLCK) and p-MLC levels in hPASMC pre-treated with 100 nM α-bt or 10 μM Me and then exposed to 15 % CSE for 24 h. Western blot representative images and protein levels quantification are shown (n = 5–7). Data are represented as the mean ± s.e.m.; Statistical comparison between groups was performed using one-way ANOVA test followed by Tukey's test (A, B, C upper) and two-sided unpaired Student's t-test (C lower) (∗)p < 0.05, (∗∗)p < 0.01, (∗∗∗)p < 0.001, (∗∗∗∗)p < 0.0001.