Figure 4: TNF-α and their receptors are upregulated in LpqH-promoted apoptosis. (a) MOs were incubated with 5 μg LpqH to induce apoptosis. Control cells were incubated with 100 μg native M. smegmatis protein (M. smeg-N). At indicated times the culture medium was collected and TNF-α production was quantitated by ELISA. (b) Culture media of apoptotic cells and control cells were collected at 60 min and TNF-α was measured by ELISA. Values are presented as the mean ± SE of three independent experiments; . (c) After 1 h incubation of cells with 5 μg LpqH, the expression of TNFR1 and TNFR2 was measured by cell surface ELISA. The relative fold increase compared with untreated cells (here set to 1) is shown as the mean ± SE of three independent experiments; . (d) MOs were preincubated with neutralizing mAb to human TNF-α, TNFR1, and TNFR2. Thereafter, without rinsing, 5 μg LpqH was added to cells for 1 h; apoptosis was measured as described in Materials and Methods. Results are given as the mean ± SE of three independent experiments. Statistically significant differences are indicated; ; .