FIG. 2.

Adenovirus-mediated overexpression of GRX1 and shRNA knock-down regulate dopaminergic cell death induced by paraquat. Overexpression of GRX1 by recombinant adenovirus Ad5CMV-GRX1 (1.5 MOI) and GRX1 knock-down with shRNA lentiviral particles were determined by Western blot (A). The effect of GRX1 overexpression (C and D) and knock-down (B) on the loss of mitochondrial activity (B and D) and cell death (C) induced by paraquat was determined as in Figure 1. In E, the effect of paraquat (48 h treatment) on the overexpression of GRX1 induced by Ad5CMV-GRX1 (1.5 MOI) was evaluated. Cleavage of α-fodrin (apoptotic marker) and accumulation of LC3-II (autophagy marker) induced by paraquat were also determined by Western blot in whole cell lysates (F). Control adenovirus contained only the cytomegalovirus promoter (AdEmpty) and/or the green fluorescent protein gene as a reporter (AdGFP). Data in graphs represent means±SEM of four independent experiments. *p<0.05, shRNA4 and 8 vs Scramble (C), or GRX1 vs. Empty values (D). Plots in (C) represent 0.5 mM PQ treatments. Contour plots and Western blots are representative of 3–4 independent experiments. Numbers in blots (italics) represent the densitometry analyses with respect to Scramble (A), Ad-GRX1 (E), and Ad-Empty samples (F) normalized to β-actin or GAPDH.