Fig 2: Schematic diagram of the apoptosis mechanism activated by glutamine limitation in glutamine-addicted tumor cells.Glutamine deprivation or inhibition of glutamine metabolism will result in GCN2-mediated TRAIL-R2 upregulation and metabolic stress-induced FLIPL downregulation, leading to caspase-8 activation and apoptosis.

Fig 3: Altered recruitment of inhibitory components to the DISC promotes caspase-8 activation in uPA knockdown cells. (a) Time-course analysis of procaspase-8 cleavage. BJELR cells were transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scramble'). Forty-eight hours after transfection, cell populations were either left untreated or challenged with 1 μg/ml TRAIL during 1, 3 or 7 h and the percentage of cells displaying cleaved caspase-8 (‘C8') was determined by flow cytometry. Images from one representative experiment out of three independent replicates are shown. Percentage of cleaved C8-positive cells is indicated. (b) DISC composition in uPA-depleted cells. BJELR cells were transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scr'), and 48 h after transfection, cell populations were either left untreated or challenged with TRAIL (1 μg/ml) during 30 min. Immunoprecipitation of Death Receptor 5 (IP-DR5) was performed and co-immunoprecipitation of cognate DISC components, uPA and uPAR, was analyzed by western blot analysis. Immunoprecipitation using isotypic IgG1 (‘IgG1') was used as background control. (c) Surface levels of TRAIL-Rs upon uPA knockdown. BJELR cells transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scr'). Forty-eight hours after transfection, surface levels of Death Receptor 5 (‘DR5'), Death Receptor 4 (‘DR4'), Decoy Receptor 1 (‘DcR1') and Decoy Receptor 2 (‘DcR2') were analyzed by flow cytometry. Isotypic IgG1 labeling was used as control for background fluorescence in scramble- (‘IgG1 scr') and siPLAU- (‘IgG1 siPLAU') transfected cells. (d) Antiapoptotic role of DcR2. BJELR cells were transfected either with pooled siRNA targeting Death Receptor 5 mRNA (‘siDR5'), Decoy Receptor 2 mRNA (‘siDcR2') or non-targeting scramble siRNAs (‘scr'), and 48 h after transfection, cell populations were either left untreated or challenged with TRAIL (1 μg/ml) during 3 h. Apoptosis was determined as the percentage of cells with positive labeling for cleaved PARP by flow cytometry. Histograms represent the mean±S.D. of three independent biological replicates. Statistical significance was calculated by applying two-tailed, unpaired Student's t-test, *P-value<0.05; **P-value<0.005. (e) Efficiency of DR5 and DcR2 knockdown at 48 h after transfection analyzed by western blot analysis. α-Tubulin, loading control

Fig 4: Role of TRAIL-R2 in apoptosis induced by glutamine deprivation in tumor cells.A (HCT116) or B (MDA-MB468) cells stably expressing a scrambled (shSc) or a TRAIL-R2 targeting shRNA (shTRAIL-R2#1) were cultured with or without glutamine and apoptosis was assessed at the indicated times (HCT116) or 48 h (MDA-MB468). TRAIL-R2 knockdown was determined by Western blotting. Tubulin and GAPDH were used as protein-loading controls. Data are presented as mean ± SD from at least three independent experiments. **P < 0.01; ****P < 0.0001; two-way ANOVA test. Tukey’s multiple comparison test. C Procaspase-8 levels and caspase-8 activation (cC8) in HCT116 cells incubated in the presence or absence of glutamine for the indicated times. D Scrambled (Sc) or shTRAIL-R2#1 HCT116 cells were cultured in the presence or absence of glutamine for 24 h. Following this incubation, TRAIL-R2 levels, caspase-8 and caspase-3 activation, as well as procaspase-8 or procaspase-3 levels were assessed by Western blotting.

Fig 5: Role of FLIP in cell death induced by glutamine deprivation.A HCT116 (left panel) or MDA-MB468 (right panel) cells were cultured in the presence or absence of glutamine for the indicated times. Tubulin and GAPDH were used as protein-loading controls. Following these treatments, FLIPL and FLIPS levels were assessed by Western blotting. B Apoptosis was assessed in pBabe or FLIPL overexpressing HCT116 cells cultured in the presence or absence of glutamine for 48 h (left panel). Procaspase-8 levels, caspase-8 activation (cC8) and FLIPL overexpression were determined by Western-blotting after 30 h of glutamine starvation (right panel). C Apoptosis was assessed in pBabe or FLIPL-overexpressing MDA-MB468 cells cultured in the presence or absence of glutamine for 48 h. FLIPL overexpression was detected by Western blotting. In B and C data are presented as mean ± SD from at least three independent experiments. **P < 0.01; ***P < 0.001; two-way ANOVA test. Tukey’s multiple comparison test. D HCT116 cells were cultured in the presence or absence of glutamine for 16 h, with or without dimethyl a-ketoglutarate (5 mM). FLIP levels, ISR activation and TRAIL-R2 upregulation were assessed by Western blotting. In E apoptosis was assessed in HCT116 cells cultured in the presence or absence of glutamine for 48 hours, with or without dimethyl a-ketoglutarate. Data are presented as mean ± SD from at least three independent experiments. **P < 0.01; ***P < 0.001; two-way ANOVA test. Tukey’s multiple comparison test.