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. 2017 Apr 6;26(2):191–200. doi: 10.4062/biomolther.2016.276

Fig. 2.

Fig. 2.

Effects of YJI-7 on LPS-stimulated ROS production and NADPH oxidase activation in RAW 264.7 macrophages. (A) Cells were pre-treated with YJI-7 for 1 h followed by stimulation with LPS for additional 24 h. After washing with Hank’s balanced salt solution (HBSS), cells were incubated with 5-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) for 30 min. ROS production was measured by the amount of CM-H2DCFDA converted into fluorescent DCF. Data are expressed as fold change in the amount of DCF relative to that observed in cells stimulated with LPS and are expressed as the mean ± SEM (n=3). Statistical significance is indicated as follows: *p<0.05 for comparison with values observed in control cells; #p<0.05 for comparisons with values observed in cells treated with LPS. (B) Effect of YJI-7 on cell viability was assessed by MTS assay. After treatment with indicated concentrations of YJI-7 for 24 h, the cells were incubated with MTS for 2 h and cell viability was determined as described in Materials and Methods. Values represent the fold change in the amount of formazan product relative to that of control cells. Data are presented as the mean ± SEM (n=3). (C) Cells were treated with YJI-7 for 24 h in the absence or presence of DPI, a pharmacological inhibitor of NADPH oxidase, and ROS production was determined as indicated previously. Data are expressed as fold change in the amount of fluorescent DCF compared to that of control cells and are expressed as the mean ± SEM (n=3). *p<0.05 for comparison with values observed in control cells; #p<0.05 for comparison with values observed in cells treated with LPS only. (D) RAW 264.7 macrophages were treated with YJI-7 for 2 h followed by the incubation with LPS for 6 h. Cellular lysates were prepared and incubated with lucigenin (100 μM) and NADPH for 30 min. NADPH oxidase activity was then assessed as described in Materials and Methods. Values represent fold change in comparison to the cells treated with LPS and are expressed as the mean ± SEM (n=3). *p<0.05 compared with the control cells; #p<0.05 compared with cells treated with LPS only.