ER Stress-Mediated Apoptosis in Mtb Infections Figure 4. Effect of eIF2a phosphorylation on intracellular survival of Mtb H37Rv. Raw264.7 cells were pretreated for 30 min with indicated concentrations of salubrinal and then infected with Mtb H37Rv (MOI = 1) for 3 h. Salubrinal remained for the rest of the infection. (A) The cells were incubated for 48 h and Western blot analysis was performed using antibodies directed against CHOP, p-eIF2a and b-actin. DMSO alone was used as the negative control. (B) Quantification of intracellular survival of Mtb H37Rv in Raw264.7 cells pretreated for 30 min with salubrinal as described above. The cells were collected at 48 h postinfection with Mtb H37Rv and bacteria number was determined by CFU counting. The statistical significance (*P,0.05) of observed differences between salubrinal treated and untreated groups following infection with Mtb H37Rv were verified by two-tailed t-test. Data represent the mean6standard error of the mean (SEM) of values obtained in three independent experiments. doi:10.1371/journal.pone.0028531.g004 Macrophages infected with Mtb generate reactive oxygen intermediates (ROI) and reactive nitrogen intermediates [22]. Since ER stress pathway involving CHOP is important in NO- and ROS-induced apoptosis [7,23], we determined to investigate the production of NO and ROS from Raw264.7 cells infected with live Mtb and heat-killed Mtb (Fig. 6B, C). Interestingly, levels of NO were significantly higher in supernatants of live Mtb infected cells as compared to the supernatants from heat-killed Mtb infected cells (Fig. 6B). L-NAME, an inhibitor of nitric oxide synthase (NOS), inhibited NO production to a comparable extent in this experiment. However, produced NO does not affect on CHOP expression (Fig. 6D). The production of superoxide was measured with dihy- droethidium staining after Mtb infection. Live Mtb infected Raw264.7 cells produced significantly increased levels of ROS compared with heat-killed Mtb infected cells (Fig. 6C). The PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28531