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. 2019 Nov 18;51(11):137. doi: 10.1038/s12276-019-0330-3

Fig. 2. COX2 was required for TNFα-induced VSMC phenotypic changes.

Fig. 2

P4-stage VSMCs were stimulated with TNFα for 4 days, and the expression levels of COX2 and contractile marker genes were assessed by western blot analysis (a) and immunocytochemistry (b). Magnification, 40×. Bar, 100 μm. c The COX2 promoter was subcloned into the pGL3 vector and was transfected into P4-stage VSMCs (n = 3). The TNFα-induced activation of the COX2 promoter was assessed as described in the “Materials and methods” section. COX2 was ectopically expressed in P4-stage VSMCs, and the expression levels of COX2, SMA, and SM22α (d) and SM22α promoter activity (e) were examined (n = 3). f P4-stage VSMCs were pretreated with a selective COX2 inhibitor (celecoxib), and TNFα-dependent gene expression changes were examined. COX2 was silenced in P4-stage VSMCs, and TNFα-dependent gene expression (g) and the loss of contractility were evaluated (n = 3) (h). *P < 0.05 compared with the untreated group. n.s. not significant. The results are presented as the means ± SEM. One-way ANOVA and Tukey’s multiple comparison test were used to determine the P values. The asterisks indicate statistical significance (P < 0.05).