Figure 4
MA and gp120 induced the expression of various isozymes of CYP, and CYP2E1 played an important role in oxidative stress. (a and b) SVGA astrocytes were treated with or without 500 μM MA and transfected with gp120 for 6 h after transfection. Upon termination of the treatments, mRNA was isolated for its expressions for various isozymes of CYP and was measured (a) as described in Materials and Methods. Similarly, cells harvested 12 h post-treatment protein was isolated and the levels of CYPs were measured using western blotting (b), with GAPDH used as the housekeeping gene. (c and d) Primary human fetal astrocytes were treated with MA and/or gp120IIIB for 6 or 12 h and expressions of CYPs were measured at the levels of mRNA (c) and protein (d), respectively. (e–g) The effect of DAS, a selective CYP2E1 inhibitor, on ROS production (e), cell death (f), and TUNEL labeling (g) in astrocytes treated with MA and/or gp120. (h–i) The effect of small interfering RNA (siRNA)-mediated knockdown of CYP2E1 on ROS (h) and TUNEL labeling (i) in MA- and/or gp120-treated SVGA astrocytes. The ROS production and cell death were compared with untreated control that was normalized at 100%. Bar graphs show mean of at least three independent experiments. The blot is representative of three independent experiments. GAPDH was used to normalize the expressions of CYPs. The bars represent mean±S.E. of at least three independent experiments with each treatment in triplicates. The P-value ≤0.05 (*) and ≤0.01 (**) were considered statistically significant using two-tailed Student’s t-test and two-way analysis of variance (ANOVA)
