S RV-induced DNA damage and premature Epigenetic Reader Domain Senescence in lung cancer cellsAlthough our data have shown that RV-induced DNA damage is associated with increased ROS production in NSCLC cells (Fig. 4), it has yet to be determined if inhibition of ROS production using antioxidants can prevent RV-induced DNA damage and premature senescence. To this end, we pre-incubated cells with NAC prior to 
RV treatment to determine if NAC can attenuate RV-induced DNA DSBs and premature senescence in lung cancer cells. As shown in Figure 5A, our data demonstrate that pretreatment with NAC significantly inhibits the formation of RV-induced cH2AX foci in A549 and H460 cells. Furthermore, inhibitor SA-b-gal staining results show that the percentage of RV-induced premature senescent cells is substantially reduced in NAC-treated cells (Figs. 5D and 5E). Taken together, these findings strongly support the hypothesis that RV induces lung cancer cell premature senescence via ROS-mediated DNA damage.RV induces premature senescence in lung cancer cellsIt has been proposed that the induction of premature senescence is an important mechanism by which ionizing radiation and many chemotherapeutic agents exert their anticancer effects [11?3,15,17,23]. Thus, we sought to examine if low dose RV treatment induces premature senescence in NSCLC cells. Because increased SA-b-gal activity is a well-established biomarker of senescence [16], we investigated if low dose RV treatment induces premature senescence in A549 and H460 cells by SA-b-gal staining. As shown in Figure 3A, the results indicate that the number of SA-b-gal positive senescent cells is markedly increased in RV-treated versus control A549 and H460 cells. Moreover, the percentage of SA-b-gal positive cells increases with the dose of RV, suggesting that RV treatment induces premature senescence in lung cancer cells in a dose-dependent manner (Figs. 3B and 3C). We also examined the expression levels of p53 and p21, two important molecules involved in the regulation of senescence [12,15,17,35], in RV-treated NSCLC cells. Western blotting data demonstrated that the expression levels of p53 and p21 were significantly increased in RV-treated cells, compared with controlRV induces Nox5 expression in lung cancer cellsNext, we sought to determine the mechanisms by which RV induces ROS generation in cancer cells. It was reported that increased intracellular cyclic AMP (cAMP) may contribute to mitochondrial ROS accumulation [39]. Interestingly, a recent study by Park et al. has shown that RV treatment increases the levels of cAMP in mouse C2C12 cells [40]. To determine if RV alters cAMP levels and in turn induces ROS generation in lung cancer cells, we detected cAMP levels in A549 and H460 cells after different does of RV treatment. The EIA results show that RV treatment has no significant effect on cAMP levels in A549 cellsResveratrol-Induced Senescence in Cancer CellsFigure 2. Low dose RV suppresses lung cancer cell growth via an apoptosis-independent mechanism. (A) Western blot assays were performed to determine the expression of activated caspase-3 and cleaved PARP in A549 cells. Actin was used as a loading control. (B) Western blot assays were performed to determine the expression of activated caspase-3 and cleaved PARP in H460 cells. Actin was used as a loading control. doi:10.1371/journal.pone.0060065.g(Figure S1A). More interestingly, the data demonstrate that RV inhibits the levels of cAMP in H460 cells (Figure S1B). These results.S RV-induced DNA damage and premature senescence in lung cancer cellsAlthough our data have shown that RV-induced DNA damage is associated with increased ROS production in NSCLC cells (Fig. 4), it has yet to be determined if inhibition of ROS production using antioxidants can prevent RV-induced DNA damage and premature senescence. To this end, we pre-incubated cells with NAC prior to RV treatment to determine if NAC can attenuate RV-induced DNA DSBs and premature senescence in lung cancer cells. As shown in Figure 5A, our data demonstrate that pretreatment with NAC significantly inhibits the formation of RV-induced cH2AX foci in A549 and H460 cells. Furthermore, SA-b-gal staining results show that the percentage of RV-induced premature senescent cells is substantially reduced in NAC-treated cells (Figs. 5D and 5E). Taken together, these findings strongly support the hypothesis that RV induces lung cancer cell premature senescence via ROS-mediated DNA damage.RV induces premature senescence in lung cancer cellsIt has been proposed that the induction of premature senescence is an important mechanism by which ionizing radiation and many chemotherapeutic agents exert their 
anticancer effects [11?3,15,17,23]. Thus, we sought to examine if low dose RV treatment induces premature senescence in NSCLC cells. Because increased SA-b-gal activity is a well-established biomarker of senescence [16], we investigated if low dose RV treatment induces premature senescence in A549 and H460 cells by SA-b-gal staining. As shown in Figure 3A, the results indicate that the number of SA-b-gal positive senescent cells is markedly increased in RV-treated versus control A549 and H460 cells. Moreover, the percentage of SA-b-gal positive cells increases with the dose of RV, suggesting that RV treatment induces premature senescence in lung cancer cells in a dose-dependent manner (Figs. 3B and 3C). We also examined the expression levels of p53 and p21, two important molecules involved in the regulation of senescence [12,15,17,35], in RV-treated NSCLC cells. Western blotting data demonstrated that the expression levels of p53 and p21 were significantly increased in RV-treated cells, compared with controlRV induces Nox5 expression in lung cancer cellsNext, we sought to determine the mechanisms by which RV induces ROS generation in cancer cells. It was reported that increased intracellular cyclic AMP (cAMP) may contribute to mitochondrial ROS accumulation [39]. Interestingly, a recent study by Park et al. has shown that RV treatment increases the levels of cAMP in mouse C2C12 cells [40]. To determine if RV alters cAMP levels and in turn induces ROS generation in lung cancer cells, we detected cAMP levels in A549 and H460 cells after different does of RV treatment. The EIA results show that RV treatment has no significant effect on cAMP levels in A549 cellsResveratrol-Induced Senescence in Cancer CellsFigure 2. Low dose RV suppresses lung cancer cell growth via an apoptosis-independent mechanism. (A) Western blot assays were performed to determine the expression of activated caspase-3 and cleaved PARP in A549 cells. Actin was used as a loading control. (B) Western blot assays were performed to determine the expression of activated caspase-3 and cleaved PARP in H460 cells. Actin was used as a loading control. doi:10.1371/journal.pone.0060065.g(Figure S1A). More interestingly, the data demonstrate that RV inhibits the levels of cAMP in H460 cells (Figure S1B). These results.
