CTSS inhibition-induced early ROS is responsible for mitochondrial damage and the second oxidative burst.

<p>(A) For the inhibition of XO-generated early ROS, cells were pretreated with 200 μM of allopurinol for 1 h and cotreated with 20 μM of 6r for an additional 4 h. The intracellular cellular ROS level was determined using DCFH-DA and was quantified by flow cytometry. The data represent the mean ± SD of 3 independent experiments. Differences were found to be statistically significant at *<i>P</i> < 0.05 (B) After pretreatment with 200 μM of allopurinol for 1 h and cotreatment with 20 μM of 6r for an additional 24 h, the mitochondrial membrane potential was determined using JC-1 and quantified by flow cytometry. The data represent the mean ± SD of 3 independent experiments. Differences were found to be statistically significant at *<i>P</i> < 0.05 (C) Reducing the XO-generated early ROS level by allopurinol attenuated mitochondria-specific ROS generation. After pretreatment with 200 μM of allopurinol for 1 h and cotreatment with 20 μM of 6r for 24 h, the mitochondria-specific ROS level was determined using MitoSOX and quantified by flow cytometry. (D) Allopurinol treatment reduces Bax translocation and cytochrome c release in 6r-treated cells. Cells were pretreated with 200 μM of allopurinol for 1 h, and then cotreated with 20 μM of 6r for 24 h. Mitochondrial and cytosolic fractions were prepared and subjected to western blot analysis for cytochrome c, Bax, ACTIN, and COX-IV.