Figure 3

Role of NOX2 in ROS production by SSc T cells. (A) A representative gp91phox immunoblot of three independent experiments is shown. T cells were isolated from healthy controls (N) and SSc patients (SSc) and then analyzed by immunoblotting with specific antibodies. The lower panel shows the densitometric analysis from distinct experiments with five healthy controls and five SSc patients. Data are means ± standard deviation (SD). ^* P <0.05 compared to normal T cells. (B) Real time-PCR for gp91phox expression in T cells was isolated from five healthy controls and five SSc patients. Each patient and control was tested three times and the mean value used to calculate the mean of each group. Data are means ± SD. ^* P <0.05 compared to normal T cells. (C) T cells isolated from five SSc patients were treated with DPI (20 μM, 1 hour), stained with 20 μM DCFH-DA and subsequently analyzed in a plate reader fluorimeter. Each treatment was tested three times and the mean value used to calculate the mean of each group. Data are means ± SD. ^* P <0.05 compared to untreated T cells (control). (D) ROS production in SSc T lymphocytes transiently transfected with control siRNA (CTRL siRNA) or gp91phox targeted siRNA (gp91phox siRNA) for 72 hours (upper panel). Data are means ± SD of three independent experiments with cells from three distinct patients. ^* P <0.05 compared to control siRNA. Transfection efficiency was monitored by western blot (lower panel). DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; NOX2, NADPH oxidase 2; ROS, reactive oxygen species; siRNA, small interfering RNA; SSc, systemic sclerosis.