Figure 4. Effects of CPUY192018 on DSS-induced cell injury in the NCM460 cells.

(A) Protective effects of CPUY192018 against the DSS-induced cell damage. The NCM460 cells were pretreated with 10 μM CPUY192018 for 10 h then exposed to various concentrations of DSS for an additional 12 h. The cell viability was determined using the MTT assay. (B) Dose-dependent protective effects of CPUY192018 against the DSS-induced cell damage. The NCM460 cells were pretreated with 1–20 μM CPUY192018 for 10 h then exposed to 0.8 μg/mL DSS for an additional 12 h. The cell viability was determined using the MTT assay. (C) Flow cytometric analysis of the apoptotic rate. The NCM460 cells were treated with 10 μM CPUY192018 for 10 h before being exposed to 0.8 μg/mL DSS for an additional 8 h. The cells were stained with FITC-Annexin V-PI, and the apoptotic rates were detected by flow cytometry. The statistical analysis of the apoptotic rates is shown in the figure. (D) The effect of CPUY192018 on the cell cycle in the NCM460 cells. The NCM460 cells were treated with 10 μM CPUY192018 for 10 h before being exposed to 0.8 μg/mL DSS for an additional 8 h. At the end of this treatment, the cells were trypsinized, incubated with RNase, stained with propidium iodide (PI), and analyzed by flow cytometry. The statistical analysis of the ratio of the NCM460 cells in the G0/G1, S and G2/M phases of the cell cycle is shown in the figure. (E) Living Cell Microscopy. The NCM460 cells were pretreated with 10 μM CPUY192018 for 10 h and then exposed to 0.4 μg/mL DSS for an additional 6 h. After this treatment, the NCM460 cells were stained with 10 μM cH₂DCF-DA for 20 min at 37 °C and living cell fluorescence microscopy was performed. The values shown are the means ± SEM (n = 3 independent observations). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey–Kramer posttest.