recombinant trail r2 fc chimeric protein  (R&D Systems)

Journal: Cell Death & Disease

Article Title: Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

doi: 10.1038/s41419-022-05346-y

Figure Lengend Snippet: A HCT116 cells were cultured in the presence or absence of glutamine for the indicated times and TRAIL-R2 mRNA levels were measured by RT-qPCR (left panel). TRAIL-R2 and TRAIL-R1 protein levels were assessed by Western blotting (middle panel). GAPDH was used as protein-loading control. Cell surface TRAIL-R2 levels (right panel) were also analyzed in HCT116 and MDA-MB468 cell lines after 24 h of glutamine deprivation in the presence of 20 µM Q-VD-OPh, as described in Materials and Methods (MFI: geometric mean fluorescent intensity). B HCT116 cells were incubated for the indicated times in medium with or without glutamine, in the presence or absence of 1 µM A92. Western blotting was performed to determine TRAIL-R2, p-eIF2α, eIF2α and CHOP levels. C HCT116 cells stably expressing either a scrambled (Sc) or a GCN2 targeting sequence (shGCN2), were incubated for 17 h in the presence or absence of glutamine. Following this incubation, TRAIL-R2 mRNA levels were measured by RT-qPCR (left panel) and GCN2, CHOP and TRAIL-R2 protein levels were assessed by Western blotting (right panel).

Article Snippet: Anti-TRAIL-R1 (AF347), anti-TRAIL-R2 (AF631) and Recombinant TRAIL-R2-Fc chimeric protein (631-T2) were from R&D Systems (Minneapolis, USA).

Techniques: Cell Culture, Quantitative RT-PCR, Western Blot, Control, Incubation, Stable Transfection, Expressing, Sequencing

Journal: Cell Death & Disease

Article Title: Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

doi: 10.1038/s41419-022-05346-y

Figure Lengend Snippet: 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.

Article Snippet: Anti-TRAIL-R1 (AF347), anti-TRAIL-R2 (AF631) and Recombinant TRAIL-R2-Fc chimeric protein (631-T2) were from R&D Systems (Minneapolis, USA).

Techniques: Stable Transfection, Expressing, shRNA, Cell Culture, Knockdown, Western Blot, Comparison, Activation Assay, Incubation

Journal: Cell Death & Disease

Article Title: Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

doi: 10.1038/s41419-022-05346-y

Figure Lengend Snippet: 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, FLIP L and FLIP S levels were assessed by Western blotting. B Apoptosis was assessed in pBabe or FLIP L overexpressing HCT116 cells cultured in the presence or absence of glutamine for 48 h (left panel). Procaspase-8 levels, caspase-8 activation (cC8) and FLIP L 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. FLIP L 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 α-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 α-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.

Article Snippet: Anti-TRAIL-R1 (AF347), anti-TRAIL-R2 (AF631) and Recombinant TRAIL-R2-Fc chimeric protein (631-T2) were from R&D Systems (Minneapolis, USA).

Techniques: Cell Culture, Western Blot, Activation Assay, Over Expression, Comparison

Journal: Cell Death & Disease

Article Title: Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

doi: 10.1038/s41419-022-05346-y

Figure Lengend Snippet: A Apoptosis in HCT116 cells cultured for 48 h in complete medium with or without AOA in the presence or absence of non-essential amino acids (NEAA). B , C HCT116 cells were transfected either with a scrambled oligonucleotide (Sc) or with two different siRNAs targeting GCN2 (siGCN2#1 or #2). 48 hours after transfection, cells were either incubated for 16 h in the presence of absence of AOA to assess phosphorylated eIF2α, eIF2α and CHOP levels by Western blotting ( B ), or during 24 h to measure apoptosis ( C ) . D HCT116 cells were transfected and treated as described in B . TRAIL-R2 and GCN2 levels were assessed by Western blotting. GAPDH was used as protein-loading control. E HCT116 cells stably expressing scrambled, caspase-8, or TRAIL-R2 targeting shRNA, were cultured in the presence or absence of AOA, and apoptosis was measured at 24 or 48 h. Caspase 8 and TRAIL-R2 knockdown were determined by Western blotting. In A , C and E , data are presented as mean ± SD from at least three independent experiments. * P < 0.05; *** P < 0.001; **** P < 0.0001; two-way ANOVA test. Tukey’s multiple comparison test.

Article Snippet: Anti-TRAIL-R1 (AF347), anti-TRAIL-R2 (AF631) and Recombinant TRAIL-R2-Fc chimeric protein (631-T2) were from R&D Systems (Minneapolis, USA).

Techniques: Cell Culture, Transfection, Incubation, Western Blot, Control, Stable Transfection, Expressing, shRNA, Knockdown, Comparison

Journal: Cell Death & Disease

Article Title: Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

doi: 10.1038/s41419-022-05346-y

Figure Lengend Snippet: Glutamine deprivation or inhibition of glutamine metabolism will result in GCN2-mediated TRAIL-R2 upregulation and metabolic stress-induced FLIP L downregulation, leading to caspase-8 activation and apoptosis.

Article Snippet: Anti-TRAIL-R1 (AF347), anti-TRAIL-R2 (AF631) and Recombinant TRAIL-R2-Fc chimeric protein (631-T2) were from R&D Systems (Minneapolis, USA).

Techniques: Inhibition, Activation Assay

Journal: Cell Death & Disease

Article Title: Limiting glutamine utilization activates a GCN2/TRAIL-R2/Caspase-8 apoptotic pathway in glutamine-addicted tumor cells

doi: 10.1038/s41419-022-05346-y

Figure Lengend Snippet:

Article Snippet: Anti-TRAIL-R1 (AF347), anti-TRAIL-R2 (AF631) and Recombinant TRAIL-R2-Fc chimeric protein (631-T2) were from R&D Systems (Minneapolis, USA).

Techniques: