Fig. 2: TRAIL-R2/DR5 is required for IL-8 induction under glutamine-deprived conditions, whereas caspase activity is not essential.

A HCT116 cells stably expressing shScrambled, shTRAIL-R2#1, or shTRAIL-R2#2 were exposed to either glutamine-replete or glutamine-deprived conditions, as shown in the graph. IL-8 mRNA levels were quantified by RT-qPCR following 24 hours of treatment. Relative IL-8 mRNA levels were determined in comparison to those in shScrambled cells cultured under glutamine-replete conditions (left panel). TRAIL-R2/DR5 knockdown was confirmed in whole-cell extracts by western blotting (right panel). GAPDH levels were used as protein loading controls. B HCT116 cells were transfected with either a scrambled oligonucleotide (Scr) or siRNA targeting TRAIL-R1/DR4. Forty-eight hours after transfection, the cells were incubated for 24 h in complete or glutamine-deprived medium, and the IL-8 mRNA levels were assessed by RT‒qPCR. Relative IL-8 mRNA levels were determined in comparison to those in scrambled cells cultured under glutamine-replete conditions. C HCT116 cells were cultured under glutamine-replete or glutamine-deprived conditions, in the presence or absence of 20 µM Q-VD, as indicated in the graphs. IL-8 mRNA levels (left panel) were assessed by RT-qPCR, and relative IL-8 mRNA levels were determined in comparison to the glutamine-containing condition at each time point. Apoptosis (right panel) was assessed by subG1 analysis, as described in the Materials and Methods section, after 16 or 24 h of treatment, as indicated. Data are presented as mean ± SD from at least three independent experiments and were analyzed by two-way ANOVA. *P < 0.05; **P < 0.01; ****P < 0.0001.