Fig. 3

Cinobufagin-induced oxidative DNA damage leads to replication stress and activation of DNA damage response. A Representative images of γH2AX immunostaining (scale bar: 10 μm) and B Quantification of γH2AX-positive cells. Cells were treated by 100 nM CBG for the indicated times. At least 5000 cells per treatment group were analyzed. Treatment by 100 nM CBG induced a rapid, time-dependent increase in both the intensity of γH2AX staining signal and the number of γH2AX-positive cells. C Western blot. Cells were treated by 100 nM CBG for the indicated times. A time-dependent, rapid increase in levels of γH2AX and phosphorylated Chk1 and Chk2 was induced by CBG treatment. n.s.: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 vs vehicle control or NAC-treated group (n = 3)