Figure 5

TIGAR protected DNA from damage through phosphorylating ATM.

(a and b) HepG2 cells or TIGAR knockdown HepG2 cells were treated with 200 mM CoCl₂ or 2.5 μg/ml epirubicin. Fluorescence intensity of phosphorylated ATM protein was detected with a confocal microscopy. P-ATM was stained red and the nucleus was stained blue. Scale bar = 25 μm. (c) Cells were treated as described above. Protein levels of TIGAR, phosphorylated ATM or total ATM was detected with Western blot analysis. GAPDH was used as a loading control. Quantitative analysis was performed with Image J. (d) Cells were treated with 200 mM CoCl₂ or 2.5 μg/ml epirubicin and ATM inhibitor KU55933 was added in corresponding group. Expression of TIGAR, phosphorylated ATM and total ATM protein was detected with Western blot analysis. GAPDH was used as a loading control. Quantitative analysis was performed with Image J. (e) DNA damage after ATM was inhibited by KU55933 under hypoxia condition. HepG2 cells were treated with or without 200 mM CoCl₂ for 10 h and KU55933 was added 1 h before CoCl₂ treatment in corresponding group. Left, representative images of Comet assay. Right, quantification of Comet tail DNA% and tail length. (f) DNA damage after ATM was inhibited by KU55933 after epirubicin treatment. HepG2 cells were treated with or without 2.5 μg/ml epirubicin for 12 h and KU55933 was added 1 h before epirubicin treatment in corresponding group. Left, representative images of Comet assay. Right, quantification of Comet tail DNA% and tail length. Values are means ± SD from 3 independent experiments. *p <0.05, **p < 0.01, *** p < 0.001, ns p > 0.05 versus corresponding groups.