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Figure 6: PUN inhibits LPS-induced oxidative stress. (a) PUN inhibits LPS-induced ROS generation. (A) RAW264.7 cells were cultured in DMEM for 12 h and then incubated with probe DCFH2DA for 15 min. (B) RAW264.7 cells were treated with 1 μg/mL LPS for 12 h and then incubated with probe DCFH2DA for 15 min. (C) RAW264.7 cells were pretreated with 100 μM PUN for 1 h before treatment with 1 μg/mL LPS for 12 h and then incubated with probe DCFH2DA for 15 min. (D) RAW264.7 cells were pretreated with 50 μM NAC for 1 h before treatment with 1 μg/mL LPS for 12 h and then incubated with probe DCFH2DA for 15 min. (b) ROS detection was performed by a fluorescence microplate reader. RAW264.7 cells were pretreated for 1 h with PUN at indicated doses (0, 50, 100, and 200 μM) in the presence or absence of 100 μM NAC and before treatment with 1 μg/mL LPS for 12 h and then incubated with probe DCFH2DA for 15 min. (c) PUN mediated SOD1 and SOD2 mRNA expression in LPS-treated RAW264.7 cells. Cells were pretreated with PUN (25, 50, and 100 μM) and then treated to 1 μg/mL LPS for 12 h; SOD1 and SOD2 mRNA expression were detected using RT-PCR. (d) PUN inhibits LPS-induced NO overproduction; RAW264.7 cells were pretreated for 1 h with or without 100 μM PUN in the presence or absence of indicated inhibitor (LY294002, 20 μM) and inducer (insulin, 10 μM) before treatment with 1 μg/mL LPS for 12 h. NO production in the supernatant was measured using Griess reaction. Data represent the mean ± SEM of three independent experiments and differences between mean values were assessed by one-way ANOVA. , , , , , and indicate significant differences compared with the control group of indicated proteins, respectively.