Figure 4.

Rv3463 maintains the active state of Mycobacterium tuberculosis (Mtb)-infected macrophages. (A) Bone marrow-derived macrophages (BMDMs) were infected with Mtb at a multiplicity of infection (MOI) of 1 for 2 h, stimulated with or without 5 μg/ml Rv3463 for the times indicated. The cells were then lysed, and total cell lysates were separated by SDS-PAGE, followed by immunoblot analysis using antibodies against phospho-ERK1/2, phospho-p38, phospho-JNK, phospho-PI3K, phospho-Akt, phospho-IκB-α, IκB-α, and β-actin. The image is representative of three experiments showing similar results. (B,C) BMDMs infected with Mtb for 4 h were incubated with or without 5 μg/ml Rv3463 for 72 h. TNF-α and IL-12p70 in the culture supernatants were measured by ELISA. Data shown are mean ± SD (n = 3) (B). The expressions of surface molecules on the BMDMs were analyzed by flow cytometry. Histograms are representative of three experiments. Bar graphs show the percentage (mean ± SD of three experiments) for each surface molecule on F4/80⁺ cells (C). *p < 0.05, **p < 0.01 and ***p < 0.001 for Rv3463-treatment compared to infection only controls.