Fig 2: Schematic summarizing how the eIF4A inhibitor CR-31 downregulates c-FLIP expression and enhances TRAIL-mediated apoptosis in GBC cells. eIF4A, eukaryotic translation initiation factor 4A; CR-31, CR-1-31B; GBC, gallbladder cancer; TRAIL, Tumor necrosis factor related apoptosis-inducing ligand.

Fig 3: The involvement of necroptosis in the muscle inflammation and muscle weakness in CIM.a HE, immunofluorescence staining against PAX7 (green), and the TUNEL staining (red). Nuclei were counterstained with DAPI (blue). The arrowhead indicates PAX7 positive TUNEL positive satellite cell. b HE and immunofluorescence staining (green) for the expression of RIPK1, RIPK3, MLKL, phosphorylated-S345 (phospho-S345) MLKL, CFLAR, and FAS in the muscle specimens of CIM mice. a, b The arrows indicate the dying muscle fibers. Scale bars indicate 20 μm. Each histological examination was performed three times and the representative images are shown. c, d The histological scores (c) of the severity of myositis and the area of necrotic muscle fibers (d) in wild-type (WT, n = 13) or Ripk3−/− (n = 12) mice on day 14 of CIM. e, f The histological scores (e) and the area of necrotic muscle fibers (f) in WT (n = 12) or MLKL−/− (n = 13) mice on day 14 of CIM. c–f Data are presented as median ± interquartile range. Mann–Whitney U test. **p < 0.01. g The grip strength of CIM mice treated with Nec1s or the vehicle (n = 12). The treatment was started immediately after the immunization in the prophylactic group (Prophylactic Tx, n = 13), or from day 7 in the therapeutic group (Therapeutic Tx, n = 14). BW; Body weight of the mice. Data are presented as mean ± SD. Two-way ANOVA test, followed by Dunnett’s multiple comparison test. **p < 0.01. h, i The histological scores (h) and the area of necrotic muscle fibers (i) on day 21 of CIM. Vehicle; n = 12, Therapeutic Tx; n = 14, Prophylactic Tx; n = 13. Data are presented as median ± interquartile range. Kruskal–Wallis test, followed by Dunn’s test. **p < 0.01. j Proportion of TUNEL+ cells out of PAX7+ cells in the muscles of CIM on day 21. Vehicle; n = 3, Therapeutic Tx; n = 3, Prophylactic Tx; n = 3. Data are presented as mean ± SD. One-way ANOVA test, followed by Dunnett’s test. *p < 0.05. c–j Data represent two independent experiments.

Fig 4: The mitochondrial transaminase GOT2 is required for the regulation of cFLIP expression upon the activation of TRAIL-induced apoptosis.a Schematic showing the reaction by which glutamate (Glu) is converted to aKG. b 8988 T cells were treated with AOA or EGCG at the indicated concentrations for 24 h, and the lysates were subjected to immunoblotting with the indicated antibodies. c and d 8988 T cells were treated with AOA or EGCG at the indicated concentrations for 24 h and treated with TRAIL (50 ng/mL) for 4 h. Cell death was assessed by Annexin V/PI staining and flow cytometry (c), and the lysates were subjected to immunoblotting with the indicated antibodies (d). The error bars indicate the s.d. of triplicate wells from a representative experiment. e 8988 T cells were treated with AOA or EGCG at the indicated concentrations for 24 h in the presence or absence of aKG (4 mM), and the lysates were subjected to immunoblotting with the indicated antibodies. f and g 8988 T cells were treated with AOA at the indicated concentrations for 24 h in the presence or absence of aKG (4 mM) and then treated with TRAIL (50 ng/mL). Cell morphology was analyzed under an inverted microscope (magnification, 100×) (f), and cell death was assessed by Annexin V/PI staining and flow cytometry (g). The error bars indicate the s.d. of triplicate wells from a representative experiment. h 8988 T cells were treated with AOA or EGCG at the indicated concentrations for 24 h in the presence or absence of aKG (4 mM), treated with TRAIL (50 ng/mL) for 4 h and then subjected to immunoblotting with the indicated antibodies. i 8988 T cells expressing control shRNA (GFP), GOT2 shRNAs, GLUD1 shRNAs, PSAT1 shRNAs, or GPT2 shRNAs were subjected to immunoblotting with the indicated antibodies. j AKG metabolite abundances in 8988 T cells expressing control shRNA (GFP), GOT2 shRNAs, GLUD1 shRNAs, PSAT1 shRNAs, or GPT2 shRNAs. The error bars indicate the s.d. of triplicate wells from a representative experiment. k and l 8988 T cells expressing control shRNA (GFP) or GOT2 shRNAs were treated with TRAIL (50 ng/mL), and cell death was assessed by Annexin V/PI staining and flow cytometry (k). Lysates were subjected to immunoblotting with the indicated antibodies (l). The error bars indicate the s.d. of triplicate wells from a representative experiment. **P < 0.01.

Fig 5: EMT reversal with ML327 is associated with a reduction in cFLIPS and cFLIPS loss is sufficient for TRAIL sensitization(A) Western blots demonstrating a consistent reduction of cFLIPS, but not cFLIPL, levels with and without TRAIL treatment in three carcinoma cell lines after 24-hour treatment of 10 μM ML327 as compared to vehicle treatment. In SW620 cells, TRAIL appears to upregulate cFLIPS and this is blocked by ML327. Representative blots of n = 3 biologic replicates shown for all 3 cell lines. (B) Western blot of siRNA-mediated knockdown of cFLIP in HCT-116 cells treated with TRAIL (50 ng/mL). cFLIP knockdown was associated with increased caspase 8 and PARP cleavage. Representative blot of n = 3 biologic replicates shown.