G1/S phase regulatory molecules are ectopically expressed in radioresistant breast cancer cells. (A) Non-transformed human mammary epithelial cells (MCF10A) and ER-PR-HER2- human breast cancer cell lines (HCC1806, HS578, MDA-MB-231 & MDA-MB-468) were irradiated at 2 Gy. Western blots of lysates prepared from both the unirradiated (U) and the irradiated (I) cells were immunoblotted with anti-cyclin A, anti-cyclin E, anti-p21CIP1 and anti-p27KIP1 antibodies. (B) Western blot analyses of lysates prepared from various unirradiated human breast cancer cell lines (triple negative and ER-PR-HeR2+ and MCF10A) were immunoblotted with anti-cyclin D1 antibody. (C) Lentivirus containing shRNA constructs specific for CDK2 or CDK4 were used to infect human breast cancer cell lines and MCF10A cells. A pLKO.1 lentiviral vector containing a randomized shRNA sequence was used as control. After puromycin selection, the surviving cells were pooled. The lysates prepared from the infected cells were immunoblotted with anti-CDK2 or anti-CDK4 antibodies to confirm the knockdown efficiency. Equal loading was assessed by immunoblotting with a β-actin antibody.