Figure 1
Expression and silencing efficiency of adenine nucleotide translocator 2 in cancer cell lines. Expression of adenine nucleotide translocator (ANT) 2 in cancer cell lines and the silencing efficiency of ANT2 siRNA and shRNA in human breast and ovarian cancer cell lines. (a) RT-PCR analysis for detecting ANT2 mRNA expression in various human cancer cell lines. To evaluate ANT2 expression levels in the human cancer cell lines of various origins (SNU668, SNU719, SNU638, NCI-H358, A549, NCI-H889, SK-HEP-1, SNU449, SNU423, SK-OV-3, SNU8, SNU840, MCF7, MDA-MB-231 and SK-BR-3), total RNA was extracted from respective cell lines and subjected to RT-PCR using specific primers for human ANT2 or β-actin (internal control). (b) RT-PCR analysis and western blotting to detect ANT isoform expressions in breast cancer cell lines as well as a non-neoplastic breast cell line. To compare ANT isoform mRNA levels in non-neoplastic breast epithelial cell line MCF10A with other breast cancer cell lines such as MCF7 and MDA-MB-231, total RNA was extracted from the respective cell lines and subjected to RT-PCR using specific primers for human ANT1/ANT2/ANT3 or β-actin. In addition, to detect ANT protein levels, total cell extracts were used for performing western blotting with anti-ANT, anti-ANT3 and anti-β-actin antibodies. (c) RT-PCR analysis and western blotting for detecting the level of ANT2 repression mediated by ANT2 siRNA and ANT2 shRNA. To assess the extinction of endogenous human ANT2 mRNA in MCF7 and MDA-MB-231 cells due to ANT2 RNA interference, respective cell lines were transfected with ANT2 siRNAs, ANT2 shRNAs, scramble siRNA as well as scramble shRNA for 48 hours. Total RNA was then extracted from respective samples and subjected to RT-PCR using specific primers for human ANT2 or β-actin. To indirectly detect the reduction of ANT2 protein by ANT2 RNA interference, MCF7 cells were transfected with ANT2 shRNA-1, shRNA-2 and shRNA-3 as well as scramble shRNA, and 48 hours later total cell extracts were collected for performing western blotting with anti-ANT and anti-β-actin antibodies.