HIV gp120 and methamphetamine-mediated oxidative stress increases astrocyte apoptosis via cytochrome P450 2E1 Ankit Shah 1, Santosh Kumar 1, Stephen D.

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HIV gp120 and methamphetamine-mediated oxidative stress increases astrocyte apoptosis via cytochrome P450 2E1 Ankit Shah 1, Santosh Kumar 1, Stephen D Simon 2, Dhirendra P Singh 3 and Anil Kumar 1 1 Division of Pharmacology and Toxicology, School of Pharmacy, 2 Department of Informatic Medicine and Personalized Health, School of Medicine, University of Missouri-Kansas City, Kansas City, MO 64108, and 3 Department of Ophthalmology and Visual Sciences, Univ. of Nebraska Medical Center, Omaha, Supplementary Figure 1. SVGA astrocytes were treated with or without the anti-oxidant, N-acetyl Cysteine (NAC)(A) or TROLOX (B), 1 hour prior to treatments with MA and/or gp120 and the MFI for ROS was measured. The ROS production were compared with untreated control that was normalized at 100%. The bars represent mean ± SE of 3 independent experiments with each treatment in triplicates. A B

AB Supplementary Figure 2. SVGA astrocytes were treated with or without the inhibitor 1 hour prior to treatments with MA and/or gp120. The effect of CYP2D6 inhibitors, fluoxetine (A) and paroxetine (B) and CYP2B6 inhibitor, Orphenadrine (OP) (C), on ROS production by MA and/or gp120. The higher doses of the respective inhibitors significantly increased ROS production. The ROS production and cell death were compared with untreated control that was normalized at 100%. The bars represent mean ± SE of 2 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 multiple ANOVA. C

AB Supplementary Figure 3. SVGA astrocytes were transfected with 20 pmole CYP2B6 or CYP2D6 siRNA for 48 hours. The cells were then reseeded for optimal confluence and treated with MA and/or gp120. The effect of CYP2B6 (A) and CYP2D6 (B) knockdown on ROS production was measured. The specificity of siRNA on gene knockdown was confirmed with western blotting (insets). ROS production was compared with control that was normalized at 100%. Scrambled sequence of siRNA, an internal control, showed no effect on gene knockdown or ROS production. The bars represent mean ± SE of 3 independent experiments with each treatment in triplicates.