Fig. 4: Caspase-8, FADD, RIPK1 and TAK1 are required for NF-κB activation and subsequent IL-8 induction under glutamine-deprived conditions. | Cell Death Discovery

Fig. 4: Caspase-8, FADD, RIPK1 and TAK1 are required for NF-κB activation and subsequent IL-8 induction under glutamine-deprived conditions.

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. 4

HCT116 cells were transfected with either a scrambled (Scr) oligonucleotide or siRNA targeting (A, B) caspase-8 (C8), (C, D) FADD, (E, F) RIPK1 or (G, H) TAK1 for 48 hours prior to treatment. For the analysis of NF-κB transcriptional activity, the cells were transfected with 200 ng of the pSI-Check2-RLuc-NF-κB-Firefly plasmid 24 h before treatment. After 16 h of glutamine deprivation in the presence of 20 μM Q-VD, luciferase activity was measured, and relative luciferase units (RLUs) were calculated in comparison to those of scrambled cells supplemented with glutamine. Protein knockdown in whole-cell extracts was confirmed by western blotting. GAPDH and α-Tubulin levels were used as a normalization control. IL-8 mRNA levels were measured by RT-qPCR after 16 h of treatment, and the relative levels of IL-8 mRNA were normalized to those in scrambled cells treated with glutamine and Q-VD. The data are presented as mean ± SD from at least three independent experiments and were analyzed by two-way ANOVA. ns not statistically significant, **P < 0.01; ****P < 0.0001.

Back to article page