Apoptotic changes of human breast cancer cell lines transfected with adenine nucleotide translocator 2 shRNA. (a) Cell morphology analysis: MDA-MB-231 cells were transfected with scramble shRNA or adenine nucleotide translocator (ANT) 2 shRNA-1 and then examined under a phase contrast microscope after 24 hours of transfection. (b) Cell proliferation assay: after 2 or 4 days of transfection with ANT2 shRNA-1 as well as scramble shRNA, the numbers of viable cells were determined using a hemacytometer after staining dead cells with Trypan Blue. (c) ATP assay: cells were transfected with ANT2 shRNA-1 as well as scramble shRNA and then lysed to quantify total intracellular ATP levels after 24 hours of incubation. Results are tabulated in relative ATP production by normalizing luminescence units (RLU) with total protein levels. (d) Cell cycle analysis: after 24 or 48 hours of transfection with scramble shRNA and ANT2 shRNA-1, the cells were trypsinized, fixed in ethanol and stained with propidium iodide to determine DNA contents. Cell cycle distributions were analyzed by flow cytometry. (e) Apoptosis analysis: cells were transfected with specific siRNA or shRNA against ANT2, and then 48 hours later the transfected cells were stained with annexin V–fluorescence isothiocyanate (FITC) and propidium iodide (PI) for flow cytometry analysis. Data are representative of three independent experiments.