CTSS inhibition by 6r causes mitochondrial damage.

<p>(A) The mitochondrial membrane potential was determined using JC-1, and was quantified using flow cytometry. Cells were treated with 20 μM of 6r for 4 h, and then stained with JC-1 for 30 min. The JC-1 aggregated form exhibited red fluorescence, indicating high membrane potential, whereas the JC-1 monomer form exhibited green fluorescence, indicating the collapse of membrane potential. CCCP was used as a positive control. Differences were found to be statistically significant at **<i>P</i> < 0.01. N.S. denotes no significant difference. (B) The inhibition of CTSS by 6r for 24 h induced the collapse of the mitochondrial membrane potential. Differences were found to be statistically significant at *<i>P</i> < 0.05. (C) Cells were exposed to 6r at the indicated concentrations for 24 h. Mitochondrial and cytosolic fractions were prepared as described in the materials and methods section, and were subjected to western blot analysis for cytochrome c, Bax, ACTIN, and COX-IV. ACTIN and COX-4 were used as the internal control for cytosolic and mitochondrial fractions, respectively. (D) Cells were treated with 6r for the indicated concentrations, and cleaved caspase-9 fragments were analyzed with western blotting. ACTIN was shown as an internal control for semiquantitative loading in each lane.