Figure 1.

PE6 is a proinflammatory molecule of M. tb, which induces robust secretion of proinflammatory cytokines. (A–D) RAW264.7 macrophage cells were treated with various concentration of PE6 (0.5, 1 and 2 μg/ml), LPS 500 ng/ml and HI PE6 (2 μg/ml). Sandwich ELISA was used to quantify cytokine TNF-α, IL-6, IL-12, and IL-10 levels using the cell culture supernatants. LPS treatment was used as a positive control for the induction of proinflammatory cytokines. Untreated and HI PE6 treated cells were used as negative controls. Purified PE6 was treated with polymyxin B agarose beads for the removal of endotoxin, and one aliquot was digested with proteinase K, followed by heat inactivation at 100 °C for 4 h. (E, F) Three million RAW264.7 cells were seeded in a six-well tissue culture plate and left overnight for adherence. Cells were infected with recombinant M. smegmatis containing pe6 and vector alone cells at (MOI 1:10) for 24 and 48 h. TNF-α and IL-6 levels were measured using sandwich ELISA. Untreated and 0 h treated cells were used as negative controls. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison post-test. Data are representative of three independent experiments. P values <0.05 were considered as significant. *P < 0.05, **P < 0.01 and, ****P < 0.0001 vs. controls. n.s., not significant.