Fig. 3: Glutamine restriction-induced upregulation of IL-8 is dependent on NF-κB activation mediated by TRAIL-R2/DR5 signaling. | Cell Death Discovery

Fig. 3: Glutamine restriction-induced upregulation of IL-8 is dependent on NF-κB activation mediated by TRAIL-R2/DR5 signaling.

From: Activation of the integrated stress response and loss of cFLIPL under glutamine limitation induce IL-8 gene expression and secretion in glutamine-dependent tumor cells

Fig. 3

A To evaluate NF-κB transcriptional activity, HCT116 cells were transfected with 200 ng of the pSI-Check2-RLuc-NF-κB-Firefly reporter plasmid 24 h prior to treatment. Luciferase activity was assessed at the indicated time points following glutamine deprivation in the presence of 20 µM Q-VD. Relative luciferase units (RLUs) were normalized to those of control cells maintained in glutamine-replete conditions and treated with Q-VD. B HCT116 cells were transfected with scrambled (Scr) or p65-targeting siRNA for 48 h before treatment. The cells were deprived of glutamine in the presence of 20 µM Q-VD for 16 h. IL-8 mRNA levels were assessed via RT-qPCR, and relative IL-8 mRNA levels were determined in comparison to those of the scrambled cells treated with glutamine and Q-VD. p65 knockdown in whole-cell extracts was confirmed by western blotting. C To determine the role of TRAIL-R2 on NF-κB transcriptional activity following glutamine deprivation, HCT116 control and TRAILR2-KO cells were transfected as described in A. After 16 h of glutamine deprivation, TRAIL-R2 levels (right panel) and luciferase activity (left panel) were assessed. Relative luciferase units (RLUs) were calculated relative to control cells cultured in glutamine-replete conditions and treated with Q-VD. D HCT116 control and TRAIL-R2/DR5-KO cells were cultured under glutamine-replete or glutamine-deprived conditions in the presence of 20 µM Q-VD for 16 h. IL-8 mRNA levels were quantified by RT-qPCR, and relative expression was calculated in comparison to control cells maintained in glutamine-replete conditions with Q-VD treatment. E HCT116 cells were transfected with either a scrambled (Scr) oligonucleotide or a siRNA targeting GCN2. For the analysis of NF-κB transcriptional activity, cells were transfected as described in section A. Luciferase activity was assessed after 16 h of glutamine deprivation, and RLUs were normalized to those of scrambled control cells cultured with glutamine and Q-VD. GCN2 and ATF4 protein levels were assessed in whole-cell extracts by western blotting. α-Tubulin, Hsp70 and GAPDH were used as protein loading controls. Data are presented as mean ± SD from at least three independent experiments and were analyzed by two-way ANOVA. *P < 0.05; ****P < 0.0001.

Back to article page