Figure 3. Ailanthone induces DNA damage and triggers activation of the ATM/ATR pathway in Huh7 cells.
(A). Representative comet images of alkaline gel electrophoresis demonstrating DNA damage in Huh7 cells (original magnification: 200×). Huh7 cells were evaluated with a comet assay after treatment with ailanthone. DNA migration was quantified by surveying tail length (B), tail DNA% (C) and olive tail moment (D) using MetaXpress software. At least 10 cells were evaluated per sample. The results are presented as the mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control. (E) Representative Immunofluorescence images of p-H2AX (Ser139). After treatment with various concentrations of ailanthone for 48 h, the cells were incubated with antibodies against p-H2AX (Ser139) and then with an Alexa Fluor 488-conjugated secondary antibody and DAPI. Images were obtained using a laser scanning confocal microscope (original magnification: 400×). (F) Graph representing the average number of p-H2AX puncta per cell. (G) Graph representing the relative fluorescence intensity of p-H2AX. The data are expressed as the mean ± SD in triplicate from one experiment. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with the control. (H) The levels of phosphorylated and total ATM, ATR, Chk1, Chk2, and H2AX were assessed by western blot. β-actin was used as a loading control.