Fig. 5: Role of cFLIP in NF-kB activation and IL-8 induction upon glutamine deprivation.
A HCT116 cells were deprived of glutamine for the indicated time periods in the presence or absence of Q-VD (20 µM). cFLIPL and cFLIPS levels were analyzed in whole-cell extracts by western blotting. B To analyze NF-κB transcriptional activity, HCT116 pBABE-ø and pBABE-FLIPL cells were transfected with 200 ng of the pSI-Check2-RLuc-NF-κB-Firefly plasmid 24 h prior to treatment. After 16 h of glutamine deprivation with 20 µM Q-VD, luciferase activity was measured, and relative luciferase units (RLUs) were calculated in comparison to those of pBABE-ø cells treated with glutamine and Q-VD. cFLIP levels in whole-cell extracts were assessed by western blotting. C HCT116 pBABE-ø and pBABE-FLIPL cells were either deprived or not deprived of glutamine in the presence of 20 µM Q-VD for 16 h. IL-8 mRNA levels were assessed by RT-qPCR. Relative IL-8 mRNA levels were determined in comparison to those of pBABE-ø cells treated with glutamine and Q-VD. D HCT116 cells were transfected with scrambled (Scr) or a siRNA targeting cFLIPL (siFL) for 48 h prior to treatment. To test NF-κB transcriptional activity, cells were transfected as described in B. cFLIPL knockdown in whole-cell extracts was confirmed by western blotting. E HCT116 cells were transfected as described in D for 48 h prior to treatment. Cells were either deprived or not deprived of glutamine in the presence of 20 µM Q-VD for 16 h. IL-8 mRNA levels were assessed by RT-qPCR. Relative IL-8 mRNA levels were determined in comparison to those of 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. **P < 0.01; ***P < 0.001; ****P < 0.0001.
