Fig. 4.
SIRT1 regulates the acetylation of MCU at K332 and influences mitochondrial calcium influx. (A) MS/MS spectra of MCU acetyl-Lys332 peptides derived from HCT116 cells by SIRT1 inhibition. (B) Lys332 in MCU is conserved in various species. (C) CO-IP assay was performed to determine the interaction of SIRT1 and MCU. Expression of MCU was determined in SIRT1 immuonprecipitates by Western blotting in HCT116 cells (left). HA-MCU and Flag-SIRT1 were co-transfected into 293T cells, and co-immunoprecipitated by anti-Flag antibody. (D) The interaction of MCU and SIRT1 was decreased in mutant K332R of MCU. CO-IP assay was performed in HCT116 cells expressed with Flag-SIRT1 and HA-MCU or HA-MCU-K332R plasmid. (E) Representative flow cytometric analysis of mitochondrial Ca2+ signal by Rhod-2AM staining in HCT116 and DLD1, which was transfected with wild-type or K332R MCU plasmids and treated with INZ for 48 h. (F) HCT116 and DLD1 cells transfected with wild-type or K332R MCU plasmids were incubated with INZ (up to 3.2 μM) for 48 h, respectively. Then the cells loaded with 1 μM Rhod-2AM (Red), and 100 nM Mito-Tracker (Green) were observed under confocal microscopy (n = 3). Blue, DAPI. The location overlap between Rhod-2 AM and mitochondria was determined by the correlation R (Pearson's correlation coefficient). Scale bar, 10 μm. (G) JC-1 assay analysis of mitochondrial membrane potential in HCT116 and DLD1 cells with indicated treatment. Cells with low mitochondrial membrane potential were quantified. (H) Annexin V/PI assay analysis of the apoptotic cells with indicated treatment. The apoptotic cells including early apoptosis and late apoptosis were statistically presented. Bars, SD; *P < 0.05; **P < 0.01; ***P < 0.001; ns P > 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)