Fig. 8.

Increase in mitochondrial NOX4 protein level induces senescence and pro-inflammatory marker expression in VSMCs. (A) Representative brightfield microscope images of VSMCs stained for SA-β-Gal activity (upper panel) and % SA-β-Gal-positive cells (lower panel, mean ± SEM, N = 3). (B). VSMC proliferation rate was determined by BrdU incorporation (representative brightfield microscope images, upper panel) and the number of BrdU-positive cells (lower panel) was assessed as described in Methods section (mean ± SEM, N = 3). Scale is 10 μm in A and B. (C) Representative Western blots showing protein levels of replication arrest markers pCHK1 (phospho checkpoint kinase 1, Ser³¹⁷) and pp27 (phospho KIP1, Thr¹⁸⁷) in 60% confluent VSMCs. (D) Representative Western blots showing protein levels of proinflammatory makers TGFβ1 and VCAM1. β-tubulin was used as a loading control in C and D. Densitometric quantification of TGFβ1 (E) and VCAM1 protein levels normalized to β-tubulin levels (F). *P < 0.05.