CTSS inhibition-induced early ROS contributes to mitochondria-dependent apoptotic signaling.

<p>(A) Allopurinol treatment reduced 6r-induced caspase-9/-3 activation. Cells with and without allopurinol pretreatment were incubated with 20 μM of 6r for 24 h, and subjected to western blot analysis for detecting cleaved caspase-9 and caspase-3. (B) Inhibition of XO expression by siRNA silencing approach. Cells transfected only with RNAiMax was used as a negative control (siNC) and transfected with scramble negative siRNA was used as a scramble control (siSC). (C) Reducing the expression of XO by siRNA decreased 6r-induced caspase-9/-3 activation. After 48 h of siRNA knockdown of XO, cells were treated with 20 μM of 6r for 24 h and the levels of cleaved caspase-9/-3 were assessed by western blotting. (D) Allopurinol treatment reduced the number of 6r-induced apoptotic cells. After pretreatment with allopurinol for 1 h and cotreatment with 6r for 24 h, the numbers of apoptotic cells were determined by Annexin V/PI double staining and quantified by flow cytometry. Differences were found to be statistically significant at *<i>P</i> < 0.05.