Fig. 5

IL-17- and TNF-α-mediated invasion is attenuated by NOX4 siRNA in RA FLS. RA FLS were pre-incubated with 0, 10, 20, and 40 μM NOX4 inhibitor (GLX351322) for 1 h and then stimulated with or without IL-17 (10 ng/ml) and TNF-α (10 ng/ml) for 1 h. a Cell migration after 23 h was measured using a transwell chamber. After solubilization, crystal violet dye was measured to determine migration. Data represent a fold change of optical density of crystal violet stained cells compared to unstimulated control cells. b Culture supernatants were analyzed by ELISA to measure secreted levels of VEGF. c RA FLS were transfected with NOX4 siRNA. After 24 h, NOX4 levels were assessed by RT-PCR and western blot. d Following NOX4 inhibition, cells were stimulated with or without IL-17 (10 ng/ml) and TNF-α (10 ng/ml) for 1 h. Cell invasion was evaluated after 3 days with a Matrigel-coated transwell chamber. Representative transwell chambers with crystal violet-stained cells are shown. Scale bar, 100 μm. Magnification is × 100. e After solubilization, crystal violet dye was measured. Data represent a fold change of optical density of crystal violet stained cells compared to control. f PI3Kδ activation were assessed in RA FLS at the indicated time points following treatment with IL-17 (10 ng/ml) and TNF-α (10 ng/ml). g RA FLS were pre-incubated with 40 μM NOX4 inhibitor (GLX351322) for 1 h and then stimulated with or without IL-17 (10 ng/ml) and TNF-α (10 ng/ml) for 1 h. After 48 h, cells were harvested and measured the expression of PI3Kδ. Data represent one experiment, which was performed in triplicate with similar results. GAPDH was used as a loading control. The bar represents the mean. *p < 0.05