ER Stress-Mediated Apoptosis in Mtb Infections infected Raw264.7 cells. Raw264.7 cells were infected with Mtb at a MOI of 1 for 3 h, and then incubated for 0–48 h. The statistical significance (***P,0.001) of observed differences between z-VAD-fmk treated and untreated groups following infection with Mtb H37Rv were verified by two- tailed t-test. Immunoblot analysis was performed as described in Materials and Methods. doi:10.1371/journal.pone.0028531.g001 Live mycobacteria induce ER stress molecules Since only live Mtb induced apoptosis when directly compared to the dead mycobacteria [21], we hypothesize that only live Mtb can induce ER stress responses. To examine whether dead bacteria could be responsible for inducing ER stress in macrophages, Raw264.7 cells were treated with live or heat- killed Mtb for 48 h. Cell lysates were examined for ER stress sensor molecules such as CHOP, BiP, and p-eIF2a. CHOP expression was induced by treatment with live, but not heat- killed, Mtb (Fig. 6). In contrast, BiP and p-eIF2a expression was Figure 2. Time-course analysis mRNA for ER stress-related genes after Mtb H37Rv infection. Raw264.7 cells were infected with Mtb H37Rv (MOI = 1) for 3 h, and then incubated for 0–6 h. (A) XBP-1 mRNA splicing was determined by RT-PCR using specific primers that was used to amplify products of unspliced and spliced mRNA. The results represent the ratio of XBP-1 splicing to XBP-1 unsplicing (XBP-1S/U ratio). (B, C) PCR amplification with the primer pair corresponding to the BiP and CHOP mRNA was performed at the indicated time points. The results were quantified by densitometry. For a positive control, cells were treated with 2.5 mg/mL tunicamycin (Tm) for 6 h. The statistical significance (*P,0.05, **P,0.01 and ***P,0.001) of observed differences between Mtb H37Rv infected and uninfected groups were verified by two-tailed t-test. Representative data from three independent experiments are shown. doi:10.1371/journal.pone.0028531.g002 PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e28531