Fig. 3: DAG-induced DNA damage occurs in S phase of the cell cycle.

a A549 cells were treated with 5 or 25 μM VAL-083 for 24 h. After that, cells were collected for cell cycle analysis using PI staining by flow cytometry. b After treatment with different concentrations of VAL-083 (1, 2.5, 5, or 10 μM) in H1792 cells for 48 h, cell cycle analysis with PI staining was performed using flow cytometry. c A549 cells were synchronized by serum starvation (ST) for 24 h before treatment with or without 5 μM VAL-083 for the indicated time periods (1, 4, 19, 24, 44, or 49 h). Cells were then collected, fixed, and subsequently stained with PI for cell cycle distribution analysis by flow cytometry. For additional experimental details, see “Materials and methods”. The representative flow cytometric plots from two individual experiments are shown. d Flow chart outlining the experimental conditions used in e. e A549 cells (synchronized by 24 h serum starvation) were treated with 50 μM VAL-083 for 1 h and replaced with complete medium for another 24 h incubation. Then, cells were fixed, permeabilized, and immunostained with anti-cyclin A2 and anti-ɣH2AX antibodies. Representative IF images are shown with cyclin A2 in green and ɣH2AX in red. The scale bar represents 10 μm. f In all, 100–120 cells were examined from each treatment condition as in e. The percentages of two categories (cyclin A2−/ɣH2AX− and cyclin A2+/ɣH2AX+) of A549 cells are shown with corresponding statistical analysis (*p ≤ 0.05; **p ≤ 0.01; Student’s t test). g A549 cells were seeded with either serum-deprived or complete medium in 96-well culture plates. After 24 h incubation, cells were treated with 0, 100 nM, 500 nM, 1 μM, 2.5 μM, 5 μM, 10 μM, 25 μM, 50 μM, or 100 μM VAL-083 for 72 h. Following the treatment, the percentage of survival cells compared to the untreated condition was determined by crystal violet assay. The data are presented as mean ± standard error. H1792 and H2122 cells were also tested in the same experimental condition