Fig. 3

A35 induces DNA breakage in M-phase cells firstly due to mitosis inhibition by targeting top2α. a K562 cells and HepG2 cells were incubated with 2 μM A35, and samples were collected at the indicated time points to detect DNA damage-associated proteins by immunoblot. b HCT116 cells were treated with the indicated concentrations of A35 for 1 h or 24 h, and cells were fixed and stained by a FITC-labeled Ser139-phosphorylated H2AX (γ-H2AX) antibody. Representative images were acquired by a confocal microscope. c K562 cells were treated with 0.5 μM A35, and γ-H2AX-positive cells were detected at various time points by a confocal microscope. d K562 cells were treated with 2 μM A35, and γ-H2AX-positive cells were detected by flow cytometry at the indicated time points. γ-H2AX-positive polyploidy cells were counted (e). a: Percentage of γ-H2AX-positive cells among total cells. b: Percentage of γ-H2AX-positive G2 cells among total γ-H2AX-positive cells. *P < 0.05; ** P < 0.01