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. 2021 Jan 18;11:622074. doi: 10.3389/fphar.2020.622074

FIGURE 5.

FIGURE 5

Koumine (KM) prevents NF-κB activation in BMDMs stimulated by LPS. BMDMs were pretreated with various concentrations of KM for 1 h and then incubated with LPS (300 ng/ml) for 1 h. (A,B) The protein levels of total p65, IκBα and their phosphorylated forms (p-p65 and p-IκBα) were analyzed by western blotting. (C,D) p65 nuclear translocation was determined by Western Blot. Mean ± SD of three independent experiments are shown. #p < 0.05 vs. vehicle group, *p < 0.05 vs. LPS group. (E) The nuclear location of p65 in BMDM macrophages was visualized by immunofluorescence analysis with an anti-p65 (green) antibody. The nuclei (blue) were stained with Hoechst 33324. KM, 200 μM, Scale bars, 50 μm. (F) BMDMs were pretreated with KM for 1 h and then incubated with LPS (300 ng/ml) for 3 or 12 h. The release of TNF-α and IL-6 in the culture supernatants was analyzed by ELISA. Mean ± SD of three independent experiments are shown. #p < 0.05 vs. vehicle group, *p < 0.05 vs. LPS group. (G) BMDMs were pretreated with KM for 1 h and then incubated with LPS (300 ng/ml) for 3 h and ATP (5 mM) for 1 h. The release of TNF-α and IL-6 in the culture supernatants was analyzed by ELISA. Mean ± SD of three independent experiments are shown. #p < 0.05 vs. vehicle group, *p < 0.05 vs. LPS plus ATP group.