Fig. 5
LCKi promotes genomic instability and augments PARPi induced genomic instability in ovarian cancer cells. A Single cell electrophoresis or COMET assay was used to validate and independently quantify the observed DNA damage. CP70 and SKOV3 cells were treated with PP2 5 µM and/or Olaparib 3 µM for 48 h. Cells (1 × 105 Cells/ml) were then harvested and mixed with LMAgarose (1:10 V/V). 50µL of LMAgarose solution was spread on a COMET slide and subjected to single-cell gel electrophoresis. B Extent of DNA damage was estimated based on measurement of COMET tail area using Image J. C CP70 and SKOV3 cells treated with PP2 5 µM and/or Olaparib 3 µM for 48 h followed by chromosomal aberration assay analysis. Arrowheads indicate the presence of abnormalities in chromosomes including breaks, gaps, and radials. D Abnormities in chromosomes were quantified (Chromosomal break, gap, radial formation) by counting by visual observation. E CP70 and SKOV3 cells were treated with Olaparib and PP2 for 48 h. Cells were harvested, lysed, and blotted for BRCA1 and γH2AX. F CP70 Parental (WT), KO, and OE (in KO background) cells were treated with Olaparib in dose dependent manner for 12 days. To identify colonies, plates were stained with crystal violet and images were captured. Colonies formed were counted and plotted as percentage of control formation in the graph. G Schematic showing LCK inhibition was sufficient to induce HR deficient state and subsequent treatment with PARP inhibitor promoted cell death. 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