When HeLa cells transiently expressing mitochondrial cox8-mSA-FLAG were treated for 12 h with 1 (0-30 µM), we found that 10 µM and 30 µM 1 successfully reduced the mSA level with a DC50 value of 4.8 µM (Figure ). A significant reduction of mSA was also observed upon 4 h treatment with 10 µM 1 (Figure ). These results indicate that 1 reduces mSA in mitochondria in a dose-and timedependent manner. We also performed similar experiments using MCF7 cells transiently expressing mitochondrial cox8-mSA-FLAG. After12 h treatment with 1 µM 1, we observed a significant decrease of mSA abundance, and the DC50 value was 0.96 µM. In the case of treatment with 3 µM 1, the mSA To confirm the involvement of ClpP in the mSA-degradation-inducing activity of 1, we carried out ClpP RNAi experiments in HeLa cells. The cells were first transfected with siRNA and incubated for 12 h, then transfected with mSA and treated with 1. Western blot analysis showed that ClpP RNAi suppressed the 1-induced degradation of mSA (Figure , Figure ). We also examined whether proteasomes and lysosomes are involved in 1-induced mSA degradation. Pharmacological inhibition of proteasome or lysosome did not affect the mSA degradation induced by 1 (Figure ). These results suggest that the 1-induced degradation of mSA depends on ClpP. Under conditions of proteasome inhibition, the intensity of the lowest-molecular-weight band related to mSA was increased, and this result is consistent with the idea that this band is due to mis-localized and/or mis-cleaved cox8-mSA-FLAG at least partially located in the cytosol. To investigate the impact of compound 1 on total cellular protein abundance, proteomics analysis was conducted on cells treated with compound 1 (Figure ). LC-MS/MS analysis detected 3105 proteins, among which 26 proteins (0.8%) were significantly decreased . Of these, 77% (20 proteins) were mitochondria-localized proteins, supporting the idea that compound 1 functions within mitochondria (Table ). Mitochondrial ClpP activator TR57, which shares the same pharmacophore as TR79, has been reported to decrease approximately 8% of detected proteins (686/~8000) in SUM159 cells. Though these findings are not directly comparable, they suggest that compound 1 may reduce target proteins more selectively than ClpP activators.