FIGURE 8.

Effect of NAC on MIOX- and HG-induced apoptosis and expression of Bax, Bcl2, and cleaved caspase-3 and activity of caspase-3. A–C, cells under HG ambience or transfected with MIOX-pcDNA had increased expression of the apoptogenic protein Bax (A (a), lanes 2 and 4 versus lane 1). The increase was augmented following concomitant treatment with HG and MIOX transfection (A (a), lane 5), and NAC treatment reduced the expression of Bax (A (a), lanes 3 and 6). Expression of anti-apoptogenic protein Bcl2 decreased with HG treatment as well as by MIOX transfection (A (b)). The decrease was augmented with the concomitant treatment with HG and MIOX transfection (A (b), lane 5), and it was restored by NAC treatment (A (b), lane 6). Band densities normalized to respective β-actin band densities of Western blots are shown in bar graphs in B and C. D–F, both HG treatment and MIOX transfection induced an increased expression and activity of cleaved caspase-3 (D (a), lanes 2 and 4 versus lane 1; F, columns 2 and 4 versus column 1). They were further increased with concomitant treatment with HG and transfection of MIOX (D (lane 5) and F (column 5)). NAC treatment reduced the expression and activity of cleaved caspase-3 (D (lanes 3 and 6) and E (columns 3 and 6)). Band density normalized to respective β-actin band density is included in E. G, no significant apoptosis was seen under LG ambience in cells transfected with EV (G (a)). MIOX transfection or HG ambience induced a moderate degree of apoptosis (G (b and c)). Apoptosis was accentuated following MIOX-pcDNA transfection under HG ambience (G (d)). Apoptosis was reduced with NAC treatment (G (e and f)), suggesting that ROS modulate the events related to apoptosis. Band density normalized to respective β-actin band density of various blots and caspase-3 activity, compared with their respective controls, is shown in bar graphs in B, C, E, and F (n = 4; *, p < 0.01; #, p < 0.05).