Figure 6.

STAT3 signaling is important for Rv2145c-mediated intracellular survival and IL-10 production. (A-F) BMDMs pretreated with pharmacological inhibitors of STAT3 (S31-201, 5 μM) for 1 h were infected with (A, C, E) Mtb at an MOI of 1 for 4 h and treated with 10 μg/ml Rv2145c or (B, D, F) Ms_vector or Ms_Rv2145c at an MOI of 10 for 4 h. Then, the cells were cultured in the presence and absence of S31-201 for the indicated times. (A, B) Total cell lysates were separated by SDS–PAGE, followed by immunoblot analysis using antibodies against phospho-STAT3, STAT3, phospho-STAT1, STAT1, iNOs and β-actin. This image is representative of three experiments showing similar results. (C, D) Bacterial CFUs were determined at the indicated times. (E, F) IL-10, IL-6, and TNF-α levels in the culture supernatants at 72 h (E) or 24 h (F) were measured by ELISA. (G–J) BMDMs were transfected with STAT3 siRNA (siSTAT3) or non-specific siRNA as a control (siCON) and were infected with (G, I) Mtb at an MOI of 1 for 4 h, and incubated with 10 μg/ml Rv2145c or (H, J) Ms_vector or Ms_Rv2145c at an MOI of 10 for 4 h, incubated for the indicated times. (G, H) Intracellular bacterial growth was determined by plating the cell lysates on 7H10 agar. (I, J) IL-10, IL-6, and TNF-α levels in the culture supernatants at 72 h (I) or 24 h (J) were measured by ELISA. *p < 0.05, **p < 0.01, and ***p < 0.001 for treatment compared with medium controls (MC) or for the difference between treatment data. n.s., no significant difference. SC, solvent controls.