Fig. 2

DOT1L is required for homologous recombination-mediated DNA DSB repair. a SW837 cells were transfected either mock or with DOT1L siRNA (SmartPool) for 48 h, treated with ionizing radiation (6 Gy), and total protein lysates were harvested at the indicated time points and immunoblotted with the indicated antibodies. H2B and HSC70 were used as loading controls. b DOT1L knockdown efficiency in siRNA-transfected SW837 cells (parallel to a) was verified in whole cell lysates with DOT1L and H3K79me3 antibodies. c Colony formation assays were performed with control and DOT1L-depleted SW837 cells from b with the indicated doses of irradiation (Gy) and data represent the mean values from the surviving fraction (SF), n = 3, ± SD, p-value (0.005–4 Gy). d HeLa cells harboring single copies of an NHEJ repair substrate (pEJ) or an HR repair substrate (pGC) were transfected with either mock or DOT1L siRNA #1 or #4 and a DSB was induced by transfecting cells with an I-SceI-expression vector (pCMV-I-SceI-3xNLS). After 48 h, the percentage of GFP-positive cells was measured using flow cytometry analysis as an indication for HR and NHEJ efficiency. e HCT116 cells with an integrated GFP reporter system for HR efficiency were transfected with the indicated siRNAs and GFP-positive cells were measured using flow cytometry analysis. f SW837 cells were transfected as in b and 72 h after transfection treated with NCS (100 ng/ml) for the indicated time points. Chromatin fractions were harvested and subjected to Western blot analysis for the indicated proteins