Figure 3.

Rv2145c induces macrophage activation via STAT3, MAPK, and NF-κB. BMDMs stimulated with Rv2145c for the indicated times were lysed, and the proteins in the total cell lysate were separated by SDS-PAGE followed by immunoblot analysis using antibodies against (A) phospho-STAT3 and (B) phospho-ERK1/2, phospho-p38, phospho-JNK, phospho-IκB-α, IκB-α, and β-actin. This image is representative of three experiments showing similar results. (C) BMDMs were plated in covered glass chamber slides and treated with Rv2145c for 1 h, and the immunoreactivity of the p65 subunit of NF-κB in cells was determined by immunofluorescence. Scale bar, 10 μm. (D, E) BMDMs were pretreated with pharmacological inhibitors of STAT3 (S31-201, 5 μM), ERK (U0126, 10 μM), p38 (SB203580, 20 μM), JNK (SP600125, 10 μM), NF-κB (BAY11-7082, 5 μM), or DMSO (SC; solvent control) for 1 h prior to treatment with Rv2145c (10 μg/ml). After 24 h, the amounts of IL-10, TNF-α, and IL-12p70 in the culture medium were measured by ELISA (D). The mean ± SD is shown for three independent experiments. The expression levels of CD80 and MHC-I were analyzed by flow cytometry (E). Bar graphs show percentages (mean ± SD of three separate experiments) for each surface molecule on F4/80⁺ cells. *p < 0.05, **p < 0.01, or ***p < 0.001 for each inhibitor treatment compared with Rv2145c-treated controls. MC, medium controls; SC, solvent controls.