Fig. 4

CD317 regulates serum deprivation-induced apoptosis through mitochondria-AIF axis. a Flow cytometry in combination with JC-1 staining performed in Hela cells. NC and siR317 cells were cultured in FBS-containing or FBS-free medium. After a 24 h-incubation, cells were harvested and subjected to JC-1 staining following flow cytometry for MMP dection. Red characters indicated the percentage of cells with low ΔΨm, while blue indicating the JC-1 ratio (red/green fluorescence). b Quantitative analysis of the percentage and JC-1 ratio shown in A. *P < 0.05. c Subcellular distribution of cytochrome c (CYC) and AIF in Hela cells detected by western blot. In brief, NC and siR317 cells were cultured in the presence or absence of FBS for 24 h and collected for subcellular fractionation extraction. Lamin A/C, GAPDH, and COX IV served as the loading control for nuclear, cytoplasmic, and mitochondrial protein, respectively. d Representative microscopy of immunostained AIF in Hela cells. NC and siR317 cells were harvested after a 24-h incubation with or without FBS, and then fixed and immunostained for AIF (red) and nucleus (blue). Pictures were taken with a fluorescence microscope. Fifteen microscopic fields per culture were evaluated in a blind manner. Colocalization of AIF and nucleus was analyzed by ImagePro Plus solfware. Pearson’s correlation coefficient (R) was used as a measure of colocalization of Red signals with blue signals. Correlation plot was corresponding to the left figure. The mean correlation coefficient value (R) ± SEM of at least fifteen fields is shown on the plots. Scale bar: 20 μm