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. 2017 Mar 2;7:43798. doi: 10.1038/srep43798

Figure 1. The Rho GTPase pathway is involved in the up-regulation of cytoskeleton-related proteins in nicotine-treated VSMCs.

Figure 1

VSMCs were pretreated with α-BtX or Y27632 for 30 min and then exposed to nicotine for 24 h. (a) Fluorescent micrographs of the cells labeled with rhodamin-phalloidin for detection of F-actin. The right panel is the magnified image within the left rectangle. The nucleus was stained by DAPI. Representative images from three independent experiments were presented as red channel (F-actin), blue channel (DAPI) and merged figure. There was no signal detected when rhodamin-phalloidin was omitted (not shown). F-actin was up-regulated when VSMCs were treated with nicotine (N) compared to the control (C) and α-BtX or Y27632 pre-treatment counteracted this effect. (b) and (c) Western blot analysis of cytoskeletal proteins, differentiation markers and SM-specific contractile proteins (α-actin, β-actin, MLC, p-MLC, desmin, calponin and caldesmon). GAPDH was used as a reference gene. In the histogram, the fold change of the control (C) was standardized to 1 compared to the nicotine-treated group. The inhibitors attenuated the effect of nicotine by approximately 50% (*p < 0.05). The results are expressed as the mean ± SEM (n = 5). (d) and (e) Activation assay of Rho GTPases in VSMCs after nicotine treatment. The results showed that α-BtX or Y27632 inhibited the activities of Rho GTPases compared to the nicotine-treated group (N). The results are expressed as the mean ± SEM (*p < 0.05, n = 5). (f) and (g) Western blot analysis of the intracellular downstream signaling proteins of Rho GTPases after nicotine treatment. MYPT1 is a target of RhoA. PAK1 and PI3K/AKT are common targets of RAC and CDC42. GAPDH was used as an internal reference. The phosphorylation of MYPT1, PAK1 and PI3K/AKT represented the activation of RhoA, RAC1 and CDC42 signaling. The results showed that α-BtX or Y27632 inhibited the phosphorylation of Rho-GTPase downstream effectors compared with the nicotine-treated group (N) (*p < 0.05). The results are expressed as the mean ± SEM (n = 5).