Fig. 2

IRAK-M over-expression resulted in higher bacterial load, while IRAK-M interference resulted in lower bacterial load in M. tb infected cells. Lentiviruses that over-expresses (IRAK-M-GFP-Lentivirus, OE) or interferes with (IRAK-M-RNAi-GFP-Lentivirus, KD) IRAK-M expression, were constructed. a Lentiviruses of negative control (NC) and OE were introduced into Jurkat cells. Cells were examined by light microscope and fluorescent microscope at 96 h post-transfection. More than 80% of Jurkat cells expressed GFP (200×, scale bar 50 μm). b Lentiviruses of NC and KD were introduced in U937 cells. Cells were examined by light microscope and fluorescent microscope at 96 h post-transfection. More than 90% of U937 cells expressed GFP (200×, scale bar 100 μm). c Expression of IRAK1-4 in Jurkat and U937 cells was evaluated by Western blot. d-g Bacterial load in Jurkat (d, e) and U937 cells (f, g) challenged with virulent M. tuberculosis H37Rv strain (MOI 10) were analyzed by acid-fast staining and colony forming units (CFU). Cells were infected with lentivirus for 96 h, challenged with H37Rv for 5 h and washed three times with PBS. d, f At 24 h post-infection, 1 × 10⁶ cells were resuspended in 20 μl PBS and fixed in 4% para-formaldehyde for 15 min on the slides. Acid-fast staining (AF) was performed and arrows indicated AF-positive bacteria in the cells (1000×, scale bar 10 μm). e, g For determination of CFU, at 5 and 24 h post-infection, 1 × 10⁵ cells were washed aseptically, homogenized and plated at 10-fold serial dilutions on Middlebrook 7H11 agar. CFU numbers per plate were counted to evaluate the bacterial load 3 to 4 weeks later. The bacterial load in cells of different groups was expressed as Log10 CFU ± SEM (**, p < 0.01; ***, p < 0.001)