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. 2023 Apr 14;14:1132265. doi: 10.3389/fimmu.2023.1132265

Figure 2.

Figure 2

C498 inhibits JAK/STAT and NFκB signaling pathways and downstream gene expression in mouse peritoneal macrophages. (A-E) Peritoneal macrophages were isolated and treated with DMSO or C498 at 2.5, 5, and 10 μM for 2 h, followed by stimulation with 20 ng/ml TNF-α for 10 min or 100 ng/ml LPS for 0.5 h (A, B), 20 ng/ml IL-6 (C), 50 ng/ml IFN-β and IFN-γ (D, E) for 10 min. Western blot analysis of whole cell lysates and quantitative analysis were determined with primary antibodies including anti-pSer176/180-IKKα/β and anti-IKK (A, B), anti-pTyr705-STAT3 and anti-STAT3 (C), anti-pTyr690-STAT2 and anti-STAT2 (D), anti-pTyr701-STAT1 and anti-STAT1 (E). F-N, Peritoneal macrophages were pretreated with DMSO or C498 (5 μM) for 30 min and then stimulated with 150 ng/ml LPS for additional 0, 0.5, 3, and 6 h. The relative mRNA levels of IL-1β (F), IL-6 (G), TNF-α (H), CXCL1 (I), CXCL2 (J), CXCL10 (K), CCL2 (L), CCL3 (M) and IFN-γ (N) were determined by RT-PCR. (O) Peritoneal macrophages were treated with 5 μM C498 for 0, 3, 6 and 10 h, cell lysates were harvested, and protein concentrations were determined. (P) Peritoneal macrophages were seeded in 96-well plates, cultured overnight, and treated with DMSO or a serial of C498. Cell viability was determined after 24 h. *P < 0.05 is considered significant. ns, not significant.