Irradiated breast cancer cell lines knocked down for CDK4 display increased frequencies of apoptosis. (A) Cells stably expressing pLKO.1, shCDK2 or shCDK4 were unirradiated (basal), irradiated at 2 or 4 Gy. Cells were fixed at 48 hours post-irradiation. Cells were subjected to immunostaining with anti-cleaved caspase-3 antibody (arrows), and an Alexa Fluor 488 secondary antibody; DNA was counter-stained with DAPI. Pictures were collected at 20× magnification. (B) The number of cells positively stained with cleaved caspase-3 were counted in a population of at least 287 cells and the results are shown as the proportion of positive cells between treatment and control groups. The experiments were repeated 6 times. Statistical significance was calculated by a Chi-square/Fisher exact test (*=p≤0.05; **=p≤0.01). (C) Cells stably expressing pLKO.1, or shCDK4 were unirradiated (0), irradiated at 2 or 4 Gy. Protein lysates were prepared 48 hours later and were subjected to Western blot with anti-cleaved PARP antibody. β-actin was used as a loading control. (D) The CDK4/6 inhibitor PD0332991 was added to cells stably expressing pLKO.1 (100 nM, 500 nM and 1000 nM for MCF10A, MDA-MB-231 and MDA-MB-468, respectively) and unirradiated (0), irradiated at 2 or 4 Gy. Protein lysates were prepared 48 hours later and were subjected to Western blot with anti-cleaved PARP antibody. β-actin was used as a loading control. Levels were normalized based on β-actin, and are indicated as fold-induction relative to non-irradiated pLKO.1 controls.