Fig. 2

LCK regulates DNA damage and repair. (A and B) CP70 WT, LCK KO via CRISPR/Cas9 (KO), and LCK OE on CRISPR background (OE) cells were treated with DMSO/etoposide 10 µM for 24 h, followed by an additional 24 h without etoposide. Immunofluorescence was performed to visualize γH2AX foci formation. Scale bar represents 10 µm. γH2AX foci were counted by image J software and 100 cells were counted and plotted. C and D CP70 WT, LCK KO via CRISPR/Cas9 (KO), and LCK OE on CRISPR background (OE) cells were treated with DMSO/etoposide 10 µM for 24 h. Cells were incubated in drug free media for another 24 h. Immunofluorescence was performed to visualize RAD51 foci formation. RAD51 foci in 100 cells were counted by image J software. E and G CP70 cells (LCK OE, LCK Y394F, LCK K273R, and LCK Y192F) were treated with etoposide for 24 h, followed by an additional 24 h without etoposide. Immunofluorescence was performed to visualize γH2AX and RAD51 foci formation. F and H RAD51 and γH2AX foci were counted on 100 representative cells by image J software and the quantification of foci was plotted in the graph. I CP70 cells (WT, LCK OE, LCK KO, LCK Y394F, LCK K273R, and LCK Y192F) were treated with increasing concentrations of etoposide for 48 h. Cell titer glow viability assays were performed to check cell viability. J CP70 cells (LCK, LCK Y394F, LCK K273R, and LCK Y192F in LCK knock out background) were grown on cover slips and treated with etoposide for 24 h followed by incubation for 0, 2, 4, 8 and 24 h. Cells were then subjected to immunofluorescence analysis to visualize γH2AX foci formation. γH2AX foci in 100 random cells were counted and plotted to visualize the γH2AX decay kinetics. One way ANOVA analysis was performed with Tukey's multiple comparisons test to compare different groups (* p < 0.05, ** p < 0.01, *** p < 0.001). Each experiment was repeated at least three times