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. 2015 Apr 27;290(24):14927–14944. doi: 10.1074/jbc.M115.658419

FIGURE 8.

FIGURE 8.

DNA-PK is a key upstream mediator for Chk activation. A, hPNAS-4 activates DNA-PKcs. A549 and Calu-1 cells were untreated or were transfected with pc3.1 or pc3.1-hPNAS-4 for 24 h, and then the levels of ATX and total and phosphorylated ATR and DNA-PKcs in both cells were analyzed by Western blotting. The lysates from cells treated with 10 μmol/liter etoposide (Etp) for 12 h were loaded as a positive control for activated ATR and DNA-PK. β-Actin was used as a loading control. B, the silencing effect of the siRNA targeting ATX was confirmed by Western blotting. A549 cells were untreated or transfected with 50 nmol/liter scrambled or sihATX for 24 h, and then the level of ATX was detected by Western blotting. β-Actin was used as a loading control. C, hPNAS-4 does not activate ATX. A549 cells were untreated/transfected with pc3.1/pc3.1-hPNAS-4 plasmid for 36 h or transfected with 50 nmol/liter sihATX for 24 h and then untreated/treated with 10 μm etoposide for an additional 12 h. The treated cells were collected to detect the expressions of ATX, p53, and phospho-p53 (Ser-15) by Western blotting. D, inhibition effects of ATM/ATR/DNA-PK inhibitors were confirmed by Western blotting. A549 cells were untreated or were treated with 10 μm etoposide in the presence or absence of ATM inhibitor KU60019 (5 μmol/liter), ATR inhibitor VE821 (1 μmol/liter), and DNA-PK inhibitor NU7026 (10 μmol/liter) for 24 h, and then the levels of total and phosphorylated ATM, ATR, and DNA-PKcs were detected by Western blotting. β-Actin was used as a loading control. E, effect of ATM/ATR/DNA-PK inhibitors on the phosphorylation of DNA-PK, Chk1, and Chk2. A549 cells were untreated or transfected with pcDNA3.1/hPNAS-4 for 24 h in the presence or absence of KU60019 (5 μmol/liter), VE821 (1 μmol/liter), and NU7026 (10 μmol/liter) for an additional 12 h, and then the expressions of total and phosphorylated DNA-PKcs, Chk1, and Chk2 were detected by Western blotting. β-Actin was used as a loading control. F, effect of siRNAs targeting ATM/ATR/DNA-PK on the phosphorylation of DNA-PK, Chk1, and Chk2. A549 cells were untreated or co-transfected with 50 nmol/liter siATM, siATR, and siDNA-PKcs with the pc3.1/pc3.1-hPNAS-4 plasmid for 36 h, and then the expressions of ATM, ATR, DNA-PKcs, Chk1, Chk2, and phosphorylated DNA-PKcs, Chk1, and Chk2 were detected by Western blotting. β-Actin was used as a loading control.