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(A) Analysis from Lawson et al , highlighting CFLAR as an immune evasion gene across multiple cell lines. (B) Time-course immunoblot showing <t>cFLIP</t> (FLIP L isoform) expression in wild-type <t>and</t> <t>TAK1-deficient</t> B16F10 cells treated with IFNγ and TNF for the indicated durations (0-8 hours). (C) Western blot of FLIP L expression after 4-hours stimulation with IFNγ, TNF, or their combination in WT and TAK1-deficient B16F10 cells. (D) cFLIP protein levels in WT and TAK1-deficient B16F10 cells treated with TNF (4 hours) with or without proteasome inhibitor MG132 (5 µM). (E-F) Similar cytokine-induced downregulation of cFLIP (FLIP L and FLIP s ) observed in HeLa cells (E) treated with IFNy (100 ng/mL), TNF (2 ng/mL) or/and TAKi (5 µM), and where indicated (F), in cells treated with Emricasan (5 µM). All these indicate a conserved effect of TAK1 inhibition across cell types. (G) Flow cytometry quantification of PI⁺ (dead) cells following 24-hours treatment with IFNγ and TNF in control, TAK1-deficient, cFLIP-deficient, or double-deficient B16F10 cells.
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(A) Analysis from Lawson et al , highlighting CFLAR as an immune evasion gene across multiple cell lines. (B) Time-course immunoblot showing <t>cFLIP</t> (FLIP L isoform) expression in wild-type <t>and</t> <t>TAK1-deficient</t> B16F10 cells treated with IFNγ and TNF for the indicated durations (0-8 hours). (C) Western blot of FLIP L expression after 4-hours stimulation with IFNγ, TNF, or their combination in WT and TAK1-deficient B16F10 cells. (D) cFLIP protein levels in WT and TAK1-deficient B16F10 cells treated with TNF (4 hours) with or without proteasome inhibitor MG132 (5 µM). (E-F) Similar cytokine-induced downregulation of cFLIP (FLIP L and FLIP s ) observed in HeLa cells (E) treated with IFNy (100 ng/mL), TNF (2 ng/mL) or/and TAKi (5 µM), and where indicated (F), in cells treated with Emricasan (5 µM). All these indicate a conserved effect of TAK1 inhibition across cell types. (G) Flow cytometry quantification of PI⁺ (dead) cells following 24-hours treatment with IFNγ and TNF in control, TAK1-deficient, cFLIP-deficient, or double-deficient B16F10 cells.
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(A) Analysis from Lawson et al , highlighting CFLAR as an immune evasion gene across multiple cell lines. (B) Time-course immunoblot showing cFLIP (FLIP L isoform) expression in wild-type and TAK1-deficient B16F10 cells treated with IFNγ and TNF for the indicated durations (0-8 hours). (C) Western blot of FLIP L expression after 4-hours stimulation with IFNγ, TNF, or their combination in WT and TAK1-deficient B16F10 cells. (D) cFLIP protein levels in WT and TAK1-deficient B16F10 cells treated with TNF (4 hours) with or without proteasome inhibitor MG132 (5 µM). (E-F) Similar cytokine-induced downregulation of cFLIP (FLIP L and FLIP s ) observed in HeLa cells (E) treated with IFNy (100 ng/mL), TNF (2 ng/mL) or/and TAKi (5 µM), and where indicated (F), in cells treated with Emricasan (5 µM). All these indicate a conserved effect of TAK1 inhibition across cell types. (G) Flow cytometry quantification of PI⁺ (dead) cells following 24-hours treatment with IFNγ and TNF in control, TAK1-deficient, cFLIP-deficient, or double-deficient B16F10 cells.

Journal: bioRxiv

Article Title: A TAK1 Cytokine Toxicity Checkpoint Controls Anti-Cancer Immunity

doi: 10.1101/2025.05.09.652721

Figure Lengend Snippet: (A) Analysis from Lawson et al , highlighting CFLAR as an immune evasion gene across multiple cell lines. (B) Time-course immunoblot showing cFLIP (FLIP L isoform) expression in wild-type and TAK1-deficient B16F10 cells treated with IFNγ and TNF for the indicated durations (0-8 hours). (C) Western blot of FLIP L expression after 4-hours stimulation with IFNγ, TNF, or their combination in WT and TAK1-deficient B16F10 cells. (D) cFLIP protein levels in WT and TAK1-deficient B16F10 cells treated with TNF (4 hours) with or without proteasome inhibitor MG132 (5 µM). (E-F) Similar cytokine-induced downregulation of cFLIP (FLIP L and FLIP s ) observed in HeLa cells (E) treated with IFNy (100 ng/mL), TNF (2 ng/mL) or/and TAKi (5 µM), and where indicated (F), in cells treated with Emricasan (5 µM). All these indicate a conserved effect of TAK1 inhibition across cell types. (G) Flow cytometry quantification of PI⁺ (dead) cells following 24-hours treatment with IFNγ and TNF in control, TAK1-deficient, cFLIP-deficient, or double-deficient B16F10 cells.

Article Snippet: Membranes were then probed overnight at 4°C with the following primary antibodies: CASP3 (CST, Cat#9662), cleaved CASP3 (CST, Cat#9661T), CASP8 (CST, Cat#4927T), cleaved CASP8 (CST, Cat#9429T), CASP8, (AdipoGen, Cat#AG-20B-0057-C100), RIPK1 (CST, Cat#3493S), PARP (CST, Cat#9542T), STAT1 (CST, Cat#9172T), TAK1 (CST, Cat#5206S), CFLIP (CST, Cat#56343), A20 (Santa-Cruz, Cat#sc-166692), FADD (CST, Cat#2782), TRADD (Santa-Cruz, Cat#sc-46653), ACTIN (Proteintech, Cat#66009-1-Ig) and GAPDH (Invitrogen, Cat#437000).

Techniques: Western Blot, Expressing, Inhibition, Flow Cytometry, Control