Journal: bioRxiv

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

doi: 10.1101/2025.05.09.652721

Figure Lengend Snippet: (A) IFNγ (1 ng/mL) and/or TNF (20 ng/mL)-induced cell death in TAK1 ⁻/⁻ cells, with or without the pan-caspase inhibitor Q-VD-OPh (40µM). Data were analysed by unpaired t-test for three replicates, *p value < 0.00001. (B) Cell death assay (PI staining) of TAK1 ⁻/⁻ and TAK1 ⁻/⁻ STAT1 ⁻/⁻ B16F10 cells following IFNγ and TNF treatment for 24 hours. Data were analysed by unpaired t-test for three replicates, *p value < 0.00001. (C) Propidium iodide (PI) staining of TAK1 ⁻/⁻ , TAK1 ⁻/⁻ CASP8 ⁻/⁻ , and TAK1 ⁻/⁻ RIPK1 ⁻/⁻ B16F10 cells treated with IFNγ and TNF treatment for 24 hours. Data were analysed by unpaired t-test for three replicates, *p value < 0.00001. (D-E) Immunoblots of apoptotic markers over a time course of IFNγ and TNF treatment (0–8 hours) in WT and TAK1 ⁻/- B16F10 ⁻ cells. TAK1-deficient cells show early and enhanced cleavage of caspase-8, caspase-3, and PARP, indicating caspase-mediated apoptosis. (F) Whole-cell lysates were prepared from HeLa cells treated with IFNγ (100 ng/mL), TNF (5 ng/mL), the TAK1 inhibitor (TAKi, 5 µM), or combinations as indicated. Caspase-8 was immunoprecipitated from cell lysates and analyzed by immunoblotting for the indicated binding partners. Inputs (left panels) show total protein levels of Caspase-8, FADD, RIPK1, TRADD, FLIPL, A20, and IgG (light chain control). Immunoprecipitates (IP: Caspase-8) (right panels) reveal inducible interactions between Caspase-8 and FADD, RIPK1, and TRADD upon cytokine stimulation in the presence of TAK1 inhibition. RIPK1 recruitment to the complex is strongly enhanced by combined IFNγ+TNF treatment with TAK1i. FLIPL and A20 are also modestly enriched under these conditions. High-exposure RIPK1 blot confirms increased interaction upon co-treatment. (G) Differential Interference Contrast microscopy for HeLa cells treated with IFNγ (100 ng/mL), TNF (2 ng/mL), the TAK1 inhibitor (TAKi, 5 µM), Emricasan (5 µM) or combinations as indicated. Images are representative of 3 independent experiments.

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: Staining, Western Blot, Immunoprecipitation, Binding Assay, Control, Inhibition, Microscopy

Journal: Journal of Virology

Article Title: Zika virus inhibits cell death by inhibiting the expression of NLRP3 and A20

doi: 10.1128/jvi.01980-24

Figure Lengend Snippet: ZIKV infection inhibits A20 expression through post-transcriptional mechanisms. ( A ) RNA sequencing of cells from ZIKV infection compared with mock. Volcano plot of differentially regulated genes, with upregulated genes labeled in red as well as TNFAIP3 . Heatmaps showing the normalized RNA sequencing values of TNFAIP3 and ACTB . ( B ) qPCR analysis of TNFAIP3 transcripts in HeLa cells infected with ZIKV or stimulated with TNF. Data are presented as the mean ± SD from three independent experiments. Multiple comparisons of ordinary one-way analysis of variance (ANOVA) were used. ( C ) Immunoblotting of A20, ZIKV-NS1, and GAPDH (loading control) in HeLa cells treated with TNF, ZIKV infection, or ZIKV infection plus TNF. ( D ) Immunoblotting of A20, ZIKV-NS1, and GAPDH (loading control) in HeLa cells treated with TNF plus CHX or ZIKV infection. ( E ) Immunoblotting of A20, HPIV3-HN, and GAPDH (loading control) in HeLa cells treated with TNF plus CHX or HPIV3 infection. ( F ) Immunoblotting of HA and GAPDH (loading control) in HeLa cells stably expressing the HA-A20. ( G ) Immunoblotting of A20, HA, CASP3, GSDME, ZIKV-E protein, and actin (loading control) in HeLa cells or HeLa cells stably expressing the HA-A20 treated with TNF plus CHX or ZIKV infection. For ZIKV infection, the values of CASP3-p17 and GSDME-p34 relative to full-length proteins were presented. The data are representative of three independent experiments.

Article Snippet: The antibody against A20 (sc-166692) was from Santa Cruz.

Techniques: Infection, Expressing, RNA Sequencing, Labeling, Western Blot, Control, Stable Transfection

Journal: Journal of Virology

Article Title: Zika virus inhibits cell death by inhibiting the expression of NLRP3 and A20

doi: 10.1128/jvi.01980-24

Figure Lengend Snippet: A20 expression is not reduced by post-translational degradation in ZIKV-infected cells. ( A ) Immunoblotting of A20, ZIKV-NS1, and GAPDH (loading control) in HeLa cells infected with ZIKV or ZIKV combined with CQ or BAF. ( B ) Immunoblotting of A20, LC3, and GAPDH (loading control) in HeLa cells treated with rapamycin. ( C ) Immunoblotting of A20, ZIKV-NS1, and GAPDH (loading control) in HeLa cells infected with ZIKV or ZIKV plus MG132. ( D ) Immunoblotting of A20, ZIKV-NS1, and GAPDH (loading control) in HeLa cells treated with TNF or infected with ZIKV. ( E ) Immunoblotting of A20 in HeLa cells stably expressing the HA-A20 treated with TNF plus CHX or infected with ZIKV. ( F ) Immunoblotting of HA, Flag, and tubulin (loading control) in HEK293T cells transfected with HA-A20, Flag-ZIKV-C, or vector plasmids. ( G ) Immunoblotting of HA, Flag, and actin (loading control) in HEK293T cells transfected with HA-A20, Flag-NS5, or vector plasmids.

Article Snippet: The antibody against A20 (sc-166692) was from Santa Cruz.

Techniques: Expressing, Infection, Western Blot, Control, Stable Transfection, Transfection, Plasmid Preparation

Journal: Journal of Virology

Article Title: Zika virus inhibits cell death by inhibiting the expression of NLRP3 and A20

doi: 10.1128/jvi.01980-24

Figure Lengend Snippet: The diagram shows that ZIKV infection inhibits cell death by reducing the expression of NLRP3 and A20 compared to other viruses. The figure was generated using Figdraw.

Article Snippet: The antibody against A20 (sc-166692) was from Santa Cruz.

Techniques: Infection, Expressing, Generated