Fig. 4: DAG-induced DNA damage is repaired by homologous recombination.

a A549 cells were synchronized with 24 h serum starvation. After that, cells were incubated in complete medium with treatment of 50 μM VAL-083 for 1 h. Then, cells were washed and replaced with complete medium for an additional incubation time of 20, 24, or 48 h. Cell lysates were then extracted for western blot analysis of the sensors and effectors involved in the HR DNA damage response pathway using the following antibodies: phospho-ATM (Ser1981), phospho-Chk2 (Thr68), phospho-Chk1 (Ser345 and Ser317), phospho-RPA32 (Ser33), and ɣH2AX. Representative images are shown from three to four independent experiments. b A549 cells were synchronized with 24 h serum starvation. After that, cells were incubated in complete medium with treatment of 50 μM VAL-083 for 1 h. Then, cells were washed and replaced with complete medium for an additional incubation time of 24 h. Representative confocal images of indicated proteins (BRCA1, RPA32, Rad51, and ɣH2AX) were shown. The scale bar represents 5 μm. c Quantification of foci-positive A549 cells presented in b from 60–80 cells per condition were shown. Statistical analyses were obtained from three independent experiments (**p ≤ 0.01; ***p ≤ 0.001; Student’s t test). d A549 cells were transfected with either negative control (C) or three BRCA1-targeting siRNAs (B1, siBRCA1-2; B2, siBRCA1-15; or B3, siBRCA1-17) for 24 h. Cells were then seeded in 96-well culture plates and treated with different concentrations of VAL-083 (0, 100 nM, 500 nM, 1 μM, 1.5 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM) for 5 days. Following the treatment, crystal violet assay was performed to detect the absorbance at 560 nm wavelength. The IC₅₀ value of VAL-083 was determined by fitting a sigmoidal dose-response curve to the data using GraphPad Prism 6. The IC₅₀ values of VAL-083 in control or BRCA1-knockdown cells are presented as mean ± standard error (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; Student’s t test). Cell lysates from the control or BRCA1-knockdown cells were analyzed by western blot with antibody against BRCA1, and GAPDH was used as a loading control. The representative images are shown from three independent experiments