Fig 1: YAK577 suppresses CM-induced oxidative stress through inhibition of the NOX2/p47phox axis in VSMCs. (A) Representative dihydroethidium (DHE) fluorescence images showing intracellular ROS levels in VSMCs cultured in GM, CM, CM + YAK577, or CM + NAC. Scale bar = 100 μm. (B) Quantification of DHE fluorescence intensity in the indicated groups (n = 14, 14, 12, 13). (C–G) Representative Western blot images and corresponding densitometric analyses of NOX2, phosphorylated p47phox at Ser370, and total p47phox in VSMCs cultured in GM, CM, or CM + YAK577. NOX2 (n = 8, 7, 6), phosphorylated p47phox (n = 8, 8, 8), and total p47phox (n = 8, 8, 8) were normalized to β-actin, and the phosphorylated p47phox/total p47phox ratio (n = 12, 11, 11) was calculated to assess p47phox activation. (H) Representative DHE fluorescence images of VSMCs treated with DMSO vehicle or GSK2795039 and subsequently cultured in GM or CM. Scale bar = 50 μm. (I) Quantification of DHE fluorescence intensity in the indicated groups (n = 16, 13, 22). Data are presented as mean ± SEM. *** p < 0.001; ** p < 0.01; * p < 0.05. GM, growth medium; CM, calcification medium; NAC, N-acetylcysteine. For DHE quantification, each dot represents one quantified microscopic field. For Western blot quantification, each dot represents one independent biological experiment (not a technical replicate).