Adenine nucleotide translocator 2 shRNA regulated Bcl-2 family members and disrupted mitochondrial membrane potentials. Adenine nucleotide translocator (ANT) 2 shRNA regulated the levels of Bcl-2 family members and disrupted mitochondrial membrane potentials to induce apoptosis in MDA-MB-231 cells. (a) DNA laddering assay: cells were transfected with ANT2 shRNA-1, and then 48 hours later total genomic DNA was extracted and subjected to electrophoresis in 2% agarose gel to examine DNA fragmentation patterns. (b) Western blotting for identifying apoptotic mechanisms: cells were transfected with ANT2 shRNA-1, shRNA-2 and shRNA-3. Cytoplasmic extracts (free of mitochondria) were prepared after 24 hours of incubation for performing western blotting with anti-Bax, anti-Bcl-xL, anti-cleaved caspase 3 and anti-α-tubulin antibodies. (c) Analysis for detecting mitochondrial membrane potential: cells were transfected with ANT2 shRNA-1, and then 24 or 48 hours later the transfected cells were stained with 3,3'-diethyloxacarbocyanine. Mitochondrial membrane potentials were quantified by flow cytometry. Data are representative of three independent experiments.