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. 2016 Apr 27;6:25130. doi: 10.1038/srep25130

Figure 4. Effects of compound 20 on the LPS-induced activation of MAPK and NF-κB and expression of inflammatory genes.

Figure 4

(A,D,E,F) MPMs pretreated with vehicle or compound 20 (2.5, 5, or 10 μM) or Xanthohumol (10 μM) for 30 min followed by incubation with LPS (0.5 μg/mL) for 20 min. The protein levels of IκB, GAPDH, p-ERK, ERK, p-P38, P38, p-JNK and JNK were measured by immunoblot analysis. (B) MPMs pretreated with vehicle or compound 20 (10 μM) for 30 min followed by incubation with LPS (0.5 μg/mL) for 1 h. Cells were subjected to fluorescence microscopy (200×), displaying the NF-κB P65-stained with Cy3-labeled antibody in pink and the nuclei-stained with 4,6-diamidino-2-phenylindole in blue. (C) MPMs pretreated with vehicle or compound 20 (2.5, 5, or 10 μM) for 30 min followed by incubation with LPS (0.5 μg/mL) for 1 h. Nuclear extracts were analyzed for NF-κB activity by EMSA. (G) MPMs pretreated with vehicle or compound 20 (10 μM) for 30 min followed by incubation with LPS (0.5 μg/mL) for 6 h. Expressions of TNF-α, IL-6, IL-1β, and COX-2 were analyzed with quantitative PCR (qPCR) using specific primers and normalization against the housekeeping gene β-actin. Data are mean values (±SEM) of 3–5 separate experiments. *p < 0.05 and **p < 0.01 vs. only-LPS stimulated group. The gels were run under the same experimental conditions. Shown are cropped gels/blots (The gels/blots of 4A and 4D-F with indicated cropping lines are shown in Supplementary Figure S8).