Mycobacterial survival and detection of cytokine expression of Ms_YrbE3A, Ms_Vec, BCG_YrbE3A, and BCG_Vec in infected RAW264.7 cells. (A) Intracellular mycobacteria of Ms_YrbE3A, Ms_Vec, BCG_YrbE3A, and BCG_Vec were counted at 0, 2, 4, 8, and 24 h postinfection. (B) Detection of IL-6 expression by RAW264.7 specific to different concentrations of purified YrbE3A. ELISA of IL-6 in the supernatants of RAW264.7 cells treated with LPS (1 μg/mL) alone or with varying concentrations of recombinant YrbE3A protein (0, 2, 5, or 10 μg/mL). RAW264.7 without LPS and YrbE3A protein served as control. Data are expressed as the mean ± SD from three separate experiments. *p < 0.05, **p < 0.01 (two-tailed unpaired t-test). (C,D) ELISA was used to detect the production of TNF-α, IL-6, and IL-1β by RAW264.7 infected by Ms_YrbE3A and Ms_Vec (C) and BCG_YrbE3A and BCG_Vec (D). Significant differences (*p < 0.05, **p < 0.01, ***p < 0.001) among each treatment were analyzed by a two-way ANOVA followed by the LSD test. Each sample was repeated in triplicate.