FIG 5 .

Rv0297PGRS protein induces ROS and NO production and host cell apoptosis. (A) Induction of RAW 264.7 cell death by the recombinant Rv0297PGRS protein of M. tuberculosis. RAW 264.7 cells were incubated with different concentrations of recombinant Rv0297PGRS for 48 h, and cell mortality was assessed by alamarBlue assay. SDS (0.1%) was used as a positive control; 50 µg/ml of BSA was used as a negative control. (B) Apoptosis of RAW 264.7 cells observed upon stimulation with 20 and 40 µg/ml of recombinant Rv0297PGRS protein. Untreated, negative control; doxorubicin, positive control for induction of apoptosis; Q1-1 (annexin V negative, PI positive), dead cells; Q2-1 (double positive), late apoptosis; Q3-1 (double negative), live cells; Q4-1 (annexin V positive, PI negative), early apoptosis. (C) Percentage of apoptotic cells. Group I, early apoptotic cells; group II, late apoptotic cells; group III, total apoptotic cells (sum of early and late apoptotic cells) as calculated from groups I and II. (D) ROS generation by macrophage cells upon stimulation with recombinant Rv0297PGRS for 30 h. Eighty micrograms per milliliter of BSA was used as a negative control. Data were plotted as fold change of mean fluorescence intensity of CellROX reagent. (E) NO production by macrophage cells upon stimulation with recombinant PGRS domain of Rv0297 for 30 h. One micromolar thapsigargin was used as a positive control. Data were plotted as fold change in nitric oxide concentration. All values were represented as means ± SDs from three independent experiments. P values for *, **, ***, and ns are <0.05, <0.01, <0.001, and >0.05, respectively.