Figure 4: 14-3-3σ reduces mitochondrial mass of cancer cells.
From: The cell cycle regulator 14-3-3σ opposes and reverses cancer metabolic reprogramming
(a,b) 14-3-3σ decreased mitochondrial mass of HCT116 colorectal carcinoma cells. HCT116 14-3-3σ+/+ (WT) and HCT116 14-3-3σ−/− cells were stained with 10-nonyl acridine orange (10-NAO; Molecular Probes, Invitrogen). 10-NAO is an orange dye associated with mitochondrial cardiolipin. Therefore, 10-NAO is frequently used to evaluate total mitochondrial mass. In this experiment, 10-NAO signal was quantified using flow cytometry and is displayed as a histogram (a) and a bar graph (b). Bars represent average±95% CI; Student’s t-test, *P<0.05. (c) Restoring 14-3-3σ expression led to a significant decline in mitochondrial mass in HCT116 14-3-3σ−/− cells. Flag-14-3-3σ expression was induced in HCT116 14-3-3σ−/− cells. Noninduced cells were used as a control. All cells were stained with MitoTracker Green FM, a green fluorescent dye used to quantify functional mitochondrial mass. MitoTracker Green FM signals were analysed using flow cytometry. (d) Bar graph of data from c show the suppressive effect of 14-3-3σ on mitochondrial mass of multiple cancer cell lines. Bars represent means±95% CI; Student’s t-test, *P<0.05. Notably, the inhibitory impact of 14-3-3σ on mitochondrial biogenesis was also observed in p53-deficient cancer cells (HCT116 p53−/− and H1299). (e) 14-3-3σ decreased mitochondrial gene expression. Flag-14-3-3σ expression was induced in HCT116 14-3-3σ−/− TetR 14-3-3σ and MDA-MB-231 TetR 14-3-3σ cells. Real-time PCR was performed using specific primers for the mitochondrial genes MT-CO1 and MT-ND1. Bars represent average±95% CI; Student’s t-test, *P<0.05. (f) 14-3-3σ diminished mitochondrial DNA copy number. Flag-14-3-3σ expression was induced in HCT116 14-3-3σ−/− TetR 14-3-3σ cells. Noninduced cells were used as a control. The expression ratios of MT-CO1/PPRC1 and MT-ND1/LPL were quantitated by using real-time PCR. Bars represent average±95% CI; Student’s t-test, *P<0.05. While MT-CO1 and MT-ND1 genes are located in mitochondrial DNA, PPRC1 and LPL1 are encoded in the nucleus. Therefore, reduction of MT-CO1/PPRC1 and MT-ND1/LPL ratios reflects a decrease in mitochondrial DNA content or mitochondrial number because of 14-3-3σ.
