FIGURE 2.
PNAS-4 is activated in response to DNA damage. A, A549 lung cancer cells were treated with the indicated cisplatin, MMS, and MMC for 24 h and analyzed for the expression of hPNAS-4 by semiquantitative RT-PCR (25 cycles). GAPDH was used as a loading control. B, A549 cells were treated as described above, and the hPNAS-4 level was monitored by Western blotting. β-Actin was used as a loading control. C, down-regulation of endogenous PNAS-4 by siRNA targeting human PNAS-4 was confirmed by Western blot. D, siRNA targeting endogenous hPNAS-4 (sihPNAS-4) led to S phase release from DNA damage-induced S phase arrest in A549 cells. A549 cells were untreated or were transfected with 50 nmol/liter scrambled or sihPNAS-4 for 24 h and then treated with 10 μg/ml cisplatin for an additional 12 h. The treated cells were used for cell cycle analysis by flow cytometry. E, silencing effects of hPNAS-4 on the cell population in each phase of the cell cycle. Bars, mean; error bars, S.D. (n = 3). F, sihPNAS-4 impaired DNA damage-induced apoptosis in A549 cells. A549 cells were untreated or were transfected with 50 nmol/liter scrambled or sihPNAS-4 for 24 h and then treated with 10 μg/ml cisplatin for an additional 24 h. The treated cells were used for apoptosis analysis by flow cytometry. G, silencing effects of hPNAS-4 on apoptosis. Bars, mean; error bars, S.D. (n = 3). H, sihPNAS-4 partially attenuated DNA damage-induced up-regulation of p21 and activation of ERK and also abolished the activation of the Chk1/2-Cdc25A pathway in response to cisplatin and etoposide. A549 cells were untreated or were transfected with 50 nmol/liter scrambled or sihPNAS-4 for 24 h and then treated with 10 μg/ml cisplatin or 10 μm etoposide for an additional 12 h. The treated cells were collected to detect the expressions of p21Waf1/Cip1, Cdc25A, and total and phosphorylated ERK, Chk1, and Chk2 by Western blotting. β-Actin was used as a loading control. *, p < 0.05; ns, not significant.