Fig. 1

M. smegmatis induces strong ER stress-mediated apoptosis in macrophages. a RAW 264.7 cells were infected with M. smegmatis at multiplicities of infection (MOIs) of 1, 3, and 5 for 24 h. b RAW 264.7 cells were infected with M. smegmatis (MOI = 5) for the indicated times, and the levels of ER stress molecules were analyzed by western blotting using specific antibodies. c, d RAW 264.7 cells were infected with M. smegmatis at an MOI of 5 and incubated for the indicated times. The mRNA levels of ER stress molecules were determined by RT-PCR. e RAW 264.7 cells were infected with M. smegmatis (MOI = 5) and incubated for 0–24 h. Induction of ATF6 was analyzed by western blotting using a specific antibody. f RAW 264.7 cells were pretreated with 4-PBA (10 mM) for 1 h and infected with M. smegmatis (MOI = 5) for 24 h. Western blot analysis was performed using antibodies against ER stress molecules. As a positive control, cells were treated with TM (1 μg/mL) for 6 h. g Annexin-V/PI staining was used to evaluate apoptosis of RAW 264.7 cells during M. smegmatis infection. Quantification of the flow cytometry results in (g) for each culture condition. The percentages of apoptotic cells (sum of early and late apoptotic cells). Data are means ± SDs of three independent experiments. **p < 0.01. M. smeg, Mycobacterium smegmatis; UN, uninfected; TM, tunicamycin; STS, staurosporine; 4-PBA, 4-phenylbutyric acid