Fig. 2
From: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells
NOX5 positively regulates Src activity. a Stable overexpression of NOX5 in the indicated ESCC cell lines tested by immunoblotting. GAPDH was used as a loading control. b Total protein lysates from the vector control or NOX5-overexpressing KYSE30 (upper panel) or KYSE410 (lower panel) cells were analyzed using antibody array against 43 kinase phosphorylation sites. c Silencing NOX5 in two specific short hairpin (sh) RNA-transduced stable ESCC cell lines examined by immunoblotting. GAPDH was used as a loading control. d vector control or NOX5-overexpressing KYSE30 (right panel) or KYSE410 (left panel) cells were pretreated with 10 μM NADPH oxidase inhibitor-DPI or control solvent for 90 min, respectively, or shRNA vector or NOX5 shRNA-1, shRNA-2, were cultured under normoxic and hypoxic conditions for 24 h. The Src activity was assayed by Src activation quantitative ELISA assay. e vector control or NOX5-overexpressing KYSE30 or KYSE410 cells were treated with ROS scavenger NAC (2 mM, pretreated with 90 min) or H2O2 scavenger-PEG-catalase (400 units/ml) or control solvent, respectively, were cultured under normoxic and hypoxic conditions for 24 h. Src activity was assessed using quantitative ELISA assay. ***P < 0.001; two-tailed unpaired Student’s t-test. Error bars represent mean ± SD of five independent experiments. f NOX5 expression was associated with pSrc (Tyr419), expression in 92 primary human ESCC specimens (cohort I). Two representative specimens with low and high levels of NOX5 were shown. Magnification, ×10 as indicated. Percentages of specimens showing low or high NOX5 expression relative to the level of pSrc. Statistical differences were evaluated using the chi-square test
