Fig. 6.

Mstn KO induced mitochondrial apoptosis in HeLa cells. (A) Mstn KO altered the mitochondrial morphology in Hela cells. Cells were transiently transfected with mito-RFP for 24 h, fixed, and the mito-RFP signals were measured using confocal microscopy. Scale bar, 10 µm. Boxed region in the top image is enlarged below. Each image is representative of three independent experiments. (B) Cells were stained with JC-1 to measure the changes in the mitochondrial membrane potential and observed by fluorescence microscopy. Scale bar, 10 µm. Each image is representative of three independent experiments. (C) Representative fluorescence image of cells loaded with TMRM. Scale bar, 10 µm. Quantitative changes in the TMRM fluorescence intensity was analyzed by Image J. At least 300 cells were analyzed based on three independent experiments. Data represent the mean ± standard error of the mean. ^**P < 0.01, unpaired two-tailed t-test. (D) Representative fluorescence image of cells loaded with rhodamine 123. Scale bar, 10 µm. Quantitative changes in the rhodamine 123 fluorescence intensity were analyzed by Image J. At least 300 cells were analyzed based on three independent experiments. Data represent the mean ± standard error of the mean. ^**P < 0.01, unpaired two-tailed t-test. (E) Immunostaining of Cyt-c was observed by fluorescence microscopy. Scale bar, 10 µm. The percentage of cells that released Cyt-c into the cytoplasm was counted. At least 300 cells were analyzed based on three independent experiments. Data represent the mean ± standard error of the mean. ^***P < 0.0001, unpaired two-tailed t-test. (F) The release of mitochondrial Cyt-c was estimated by examining the Cyt-c protein content of mitochondria and the extracted mitochondria-free cytoplasmic fraction by western blotting. Prohibitin (PHB) and β-actin antibodies were used as loading controls for the mitochondrial and cytoplasmic fractions, respectively. Results are representative of three independent experiments.