Fig. 4

M1-type polarization of U937 cells induced by CpG7909 was inhibited by M. tb infection, which can be reversed by IRAK-M knockdown in U937 cells. a 2 μg/ml CpG7909 induced M1-type polarization of U937 cells. U937 cells were stimulated with CpG7909 (0, 0.5 μg/ml and 2 μg/ml) for 24 h and 50 μg of cell lysates was analysed by Western blot to detect the expression of phosphorylated ERK1/2 and iNOS. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001). b M. tb infection inhibited M1-type polarization of U937 cells induced by CpG7909. U937 cells were stimulated with CpG7909 (2 μg/ml) for 24 h, then challenged with H37Rv (MOI 10) for 24 h. Western blot was performed to detect IRAK4 and iNOS. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001). c IRAK-M knockdown rescued M1-type polarization of U937 cells induced by CpG7909, which was inhibited by M. tb infection. U937 cells were infected with IRAK-M KD lentivirus for 96 h, stimulated with CpG7909 (2 μg/ml) for 24 h, challenged with H37Rv (MOI 10) for 24 h. Fifty microgram of cell lysates was analysed by Western blot to detect IRAK4 and iNOS. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001). d IRAK-4 and IRAK-M expression in CpG-stimulated and M. tb-infected U937 cells. U937 cells grown on the coverslips were stimulated with CpG7909 (2 μg/ml) for 24 h, then infected with H37Rv (MOI 10) for 24 h, co-stained with anti-IRAK4 (Cy3) or anti-IRAK-M (Cy3) and DAPI (blue) by immunocytochemistry (400×, scale bar 50 μm)