Figure 3

GFW enhanced ROS accumulation and activation of ATM/CHK2 and ATM/P53 signaling in MB49 cells. (A) MB49 cells were pre-incubated with DCFH-DA for 1 h, followed by treatment with 2 mg/mL GFW or 50 µmol/L H₂O₂ for 3 h. Cells were then examined using flow cytometry to identify DCF fluorescent cells. Data are represented by M1 and M2 percentage of fluorescent cells. M1 is placed around the untreated cells. M2 is placed to the right of M1 as a designation of positive events. The mean ± SEM (n = 3) of percentage gated for M2 is shown. Significant differences from the untreated controls are indicated by **p ≤ 0.01 or ***p ≤ 0.001, as determined by one-way ANOVA and Dunnett comparison test. (B) MB49 cells untreated versus cells treated with GFW for 2, 4 and 6 h were examined for levels of ATM, CHK2 and Thr68-phosphorylated CHK2 by using Western blot analysis. (C) The expression levels of P21, P53 and Ser15-phosphorylated P53 were subsequently quantified in MB49 cells that did or did not undergo GFW treatment for 6 h. GAPDH protein was used as a loading control. Numbers indicate band intensities as determined by ImageJ software (National Institutes of Health; http://rsb.info.nih.gov/ij/).