Fig. 5. Binding of arsenic to Hsp60 disrupts complexation of Hsp60 with p53 and survivin, leading to degradation of p53 and survivin. (a) and (b) IP-western blots of endogenous Hsp60 associated with survivin or p53 in NB4 cells treated with ATO at different concentrations. (c) and (d) Immunolocalization of endogenous Hsp60 with p53 and survivin. Co-staining was performed using antibodies specific for Hsp60 (green), survivin (red), p53 (red) and a fluorochrome conjugated secondary antibodies against rabbit or mouse IgG. Cells treated with ATO (1.2 μM) or without (as control, upper panel) were analyzed using confocal microscopy (×63 oil lens). The images were visualized with a Zeiss microscope. Scale bars: 4 μm. (e) and (f) Arsenic mediates proteasome degradation of p53 and survivin. Treatment of NB4 cells with MG132, a 26S proteasome inhibitor prevents arsenic induced protein degradation in cells. Western blots were prepared with the extracts of NB4 cells untreated or pretreated with 25 μM MG132 for 4 h in the absence and presence of 0.8 μM As₂O₃, β-actin was used as control. Protein contents were quantified by densitometric analysis. Means ± SEM; n = 3; *p <0.05, **p <0.01 versus vehicle control. (g) Proposed the mechanism of ATO induced apoptosis through inhibition of key target Hsp60 and degradation of survivin and p53.