Fig. 2.

Inhibition of SIRT1 leads to mitochondrial dysfunction and mitophagy suppression. (A) Western blot analysis of the expression of mitochondrial dynamics-related proteins, MFN1, MFN2, Tom20, Drp1 and FIS1, in HCT116 and DLD1 cells treated with different concentration of INZ (0, 0.8, 3.2 and 12.8 μM) for 48 h. (B) Mitochondrial morphology of HCT116 and DLD1 with or without INZ treatment (3.2 μM, 48 h) was determined by confocal microscope (n = 3). Cells were labeled with Mito-tracker (red) for visualization, mitochondria in tubular, short tubular and fragment were statistically analyzed. Scale bars, 10 μm for HCT116 and 5 μm for DLD1. (C) Representative TEM images of DLD1 treated with INZ (up to 12.8 μM) for 48 h, and the mitochondrial morphology with tubular and fragment were statistically analyzed (n = 3). Scale bars, 1 μm and 2 μm. (D) Western blot determining the ubiquitin level of mitochondria from HCT116 and DLD1cells treated with INZ (for 48 h) or CCCP (for 2 h, positive control). (E) The localization of mitochondria and lysosome in HCT116 cells treated with INZ (12.8 μM, 48 h) was visualized by confocal microscope (n = 3). Green, Mito-tracker; Red, Lyso-Tracker. Scale bars, 10 μm and 20 μm. (F) Confocal microscopy determining the recruitment of LC3 to mitochondria in HCT116 with indicated treatment (n = 3). Green, LC3-GFP; Red, Mito-Tracker. Scale bars, 5 μm and 20 μm. (G, H) Effects of CCCP-induced ubiquitination of MFN2 and Tom20 with or without INZ treatment. HCT116 cells were pretreated with 12.8 μM INZ for 24 h and then treated with CCCP (10 μM) for 6 h, then the ubiquitination of MFN2 and Tom20 was analyzed. Bars, SD; **P < 0.01; ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)