Fig. 4

LCK inhibition attenuates the HR repair pathway. A Schematic of DNA repair assay in U2OS osteosarcoma cells stably transduced with the DR-GFP reporter system. This reporter system contains an upstream mutated GFP followed by a downstream truncated GFP. Transfection of I-SceI endonuclease induces double strand breaks in the upstream gene that can be repaired by the HR repair machinery leading to restored GFP expression that is quantified by fluorescence activated cell sorting (FACS). B U2OS cells were treated with 0 (DMSO) 5, 7, 10 μM of the LCKi PP2 for 48 h. Alternatively, cells were transfected with Sh Con, LCK KD1 or KD2 for 24 h and incubated in serum enriched medium for another 24 h. Cells were then subjected to DR-GFP assay followed by FACS. C Ovarian cancer cells were treated with PP2 or Olaparib for 48 h and immunoblot experiment was performed to check PARylation. D CP70 and SKOV3 cells were treated with 0 (DMSO) 5, 7, 10 μM of PP2 for 48 h then harvested, lysed, and immunoblotted for Ku70 and Ku80 protein expression. GAPDH was used as loading control. E Schematic model summarizing LCK inhibition specificity for HR DNA repair. 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)