Fig. 1.

DOT1L is required for the proper DNA damage response. a U2OS cells were transfected either mock (Control—Cont) or with siRNA (smart pool of 4 siRNA—SP) for DOT1L. After 48 h of transfection, cells were treated with neocarzinostatin (NCS; 100 ng/ml) to induce DNA DSBs for the indicated time points. Total protein lysates were analyzed by Western blot for the indicated proteins. b Similar to a, U2OS cells were transfected with siRNA and after 48 h of transfection, cells were treated with NCS for the indicated time points and processed for immunofluorescence analysis for γH2AX in red and nuclei were stained with DAPI in blue (Scale bar—10 μM). c Quantification of γH2AX and pKAP1 levels in mock or DOT1L siRNA-transfected U2OS cells treated with NCS for the indicated times from b. More than 50 cells were measured for each condition and represented as relative intensity in each cell, mean values are represented in the plot n = 3, ± SD, p values (**0.005 and *0.01). d SW837 cells were either mock (Cont) transfected or with DOT1L siRNA, after 72 h cells were treated with NCS (100 ng/ml) to induce DSBs and total protein lysates were immunoblotted with the indicated antibodies. e Proximity ligation assay (PLA) for γH2AX and H3K79me3 in SW837 cells transfected with either mock (Cont) or DOT1L siRNA for 72 h prior to treatment with NCS (100 ng/ml) for the indicated time points before processing for PLA. Red dots indicate regions of close proximity between γH2AX and H3K79me3. Nuclei were stained with DAPI (Scale bar—10 μM)