Fig. 2: DAG induces DNA damage in NSCLC cells.

a A549, H1792, and H2122 cells were treated with 20 μM VAL-083 for 24 h followed by washing and replacing with complete medium in culture for various periods of time (0, 4, 8, 24, 48, or 72 h). Then, cells were collected for protein extraction, and 50 μg was analyzed for phosphorylated and total H2AX expression by western blot using specific rabbit polyclonal antibodies as described under “Materials and methods”. Representative images are shown for the time-course effect of VAL-083 on ɣH2AX expression. Total H2AX and GAPDH served as loading controls. b Cultured A549 cells were treated with 20 μM VAL-083 for 24 h. After that, cells were washed and replaced with complete medium for an additional incubation time of 0, 4, 8, 24, 48, or 72 h. Then, cells were fixed, permeabilized, and immunostained with anti-ɣH2AX antibody. Quantification of ɣH2AX foci (cells with >10 foci were considered as “foci-positive”) from 40 to 50 cells per sample was shown. c Representative confocal images from each experimental conditions in b were shown with ɣH2AX in red. The scale bar represents 5 μm