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. 2018 Dec 28;24(1):96. doi: 10.3390/molecules24010096

Figure 8.

Figure 8

The role of ROS on MHY440-induced apoptosis in AGS cells. (A) Cells were treated with 5.0 μM MHY440 for several different times and stained with DCF-DA. Intracellular ROS levels were measured using flow cytometry. Data are means ± SD of three separate experiments. Significance was determined using Student’s t-test (*** p < 0.001 compared with vehicle-treated cells). (B) Cells were treated at various concentrations for 1 h. Data are expressed as the means ± SD of three separate experiments. Significance was determined using Student’s t-test (** p < 0.01 compared with vehicle-treated cells). (C) Cells were pretreated with or without 5 mM NAC for 1 h and then treated with 5.0 μM MHY440 for 1 h. Intracellular ROS levels were measured using fluorescence microscopy. Representative results from three independent experiments are shown. (D) Cells were treated with 5.0 μM MHY440 for 1 h after pretreatment with or without 5 mM NAC for 1 h. Data are means ± SD of three separate experiments. Significance was determined using Student’s t-test (* p < 0.05 compared with vehicle-treated cells; # p < 0.05 compared with 5.0 μM MHY440-treated cells). (E) The expression of apoptosis in cells pretreated with 5 mM NAC and 2.5 μM MHY440 was determined using PI staining and flow cytometry analysis. Data are means ± SD of three separate experiments. Significance was determined using Student’s t-test (** p < 0.01 compared with vehicle-treated cells; # p < 0.05 compared with 5.0 μM MHY440-treated cells). (F) Total cell lysates of cells treated with or without 2.5 μM MHY440 after pretreatment with or without 5 mM NAC were analyzed using western blot analysis for the expression level of PARP. β-actin was used as a loading control. Representative results from three independent experiments are shown. (G) Total cell lysates from cells treated with 2.5 μM MHY440 alone or pretreated with 5.0 mM NAC for 24 h were analyzed using western blot analysis for the following antibodies: p-ATM (Ser1981), p-ATR (Ser428), γ-H2AX (Ser139), p-Chk1 (Ser345), p-Chk2 (Thr68), and p-p53 (Ser15). β-actin was used as a loading control. Representative results from three independent experiments are shown.