Figure 4

pERK1/2 implication in ROS production. (A) T lymphocytes from five SSc patients were incubated with PD98059 (50 μM, 2 hours) or left untreated. After staining with 20 μM DCFH-DA, fluorescence was analyzed in a plate reader fluorimeter (left panel). Each treatment was tested three times and the mean value used to calculate the mean of each group. Data are means ± standard deviation (SD). ^* P <0.05 compared to untreated T cells (control). Representative histogram of ROS production by SSc T cells is shown in the right panel. Untreated (control, grey line) and PD98059 treated cells (black line) were stained with 2 μM DCFH-DA and analyzed by FACS analysis. (B) Representative pERK1/2 immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), 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 ± SD. ^* P <0.05 compared to untreated normal T cells. ^** P < 0.05 compared to untreated SSc T cells. (C) Representative Ha-Ras immunoblot of three independent experiments is shown (upper panel). T lymphocytes were isolated from five healthy controls and five SSc patients, treated with NAC (10 mM, 1 hour) or DPI (20 μM, 1 hour), 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 ± SD. ^* P <0.05 compared to untreated normal T cells. ^** P <0.05 compared to untreated SSc T cells. DCFH-DA, 2′, 7′-dichlorodihydrofluorescin diacetate; DPI, diphenylene iodonium; NAC, N-acetylcysteine; ROS, reactive oxygen species; SSc, systemic sclerosis.