Fig. 8.

Effects of SIRT inhibition and SIRT3 overexpression on the melatonin-shikonin (Mel-SHK)-induced HeLa cell cytotoxicity. (A) Immunoprecipitation analysis to demonstrate the effects of Mel and/or SHK on the direct binding of SIRT3 with SOD2. (B) apoptosis via caspase activation, (C) sub-G1 phase accumulation and (D) MMP losses were investigated in HeLa cells pre-treated with the SIRT inhibitor 4-BR before combined treatment with Mel-SHK to examine the role of SIRT3/SOD2 in Mel-SHK-induced cancer cell death. (E) SIRT3 overexpression in HeLa cells transfected with sirT3-Flag plasmid was confirmed by western blotting. (F–L) SIRT3-overexpressing HeLa cells were treated with Mel-SHK, and all parameters previously shown to be affected by Mel-SHK were evaluated. (M, N) The effect of SIRT3 overexpression on the MAPK and AKT pathways was also evaluated. Relative protein expression was calculated for all the proteins in western blots. Actin was used for normalisation and as a loading control. All experiments used SHK at 3 μM and Mel at 0–2 mM. Flow cytometry data are presented as histograms and bar graphs. One representative figure from at least three independent experiments is shown for each assay. The data in each bar graph are presented as the means ± SEM. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 relative to Mel-SHK treated cells.