Tnf α 300 01a

Actinomycin D (ActD) was purchased from Sigma-Aldrich (St. Louis, USA). Lipofectamine 2000 (11668–027) and Opti-MEM I reduced serum medium were obtained from Invitrogen (Carlsbad, USA). MK2 siRNA, HuR siRNA and negative control siRNA were received from GenePharma (Shanghai, China). TNF-α (300-01A) was purchased from PeproTech (Rocky Hill, USA). Phosphatase Inhibitor Cocktail (04906845001) was from Roche (Mannheim, Germany). An enhanced chemiluminescent (ECL) kit were obtained from Pierce (Rockford, USA). The protein assay kit, nuclear and cytoplasmic protein extraction kit (P0027) and the RIPA Lysis Buffer (P0013C) were purchased from Beyotime Institute of Biotechnology (Nantong, China). Antibodies raised against MK2 (#3042), phospho-MK2 (Thr334, #3041), ICAM-1 (#4915) was obtained from Cell Signaling (Danvers, USA); a monoclonal antibody against HuR (ab14371) was received from Abcam (Cambridge, USA); antibodies against β-actin (P30002) and histone (P30266) were from Abmart (Shanghai, China);IL-8 ELISA Kit (BMS204/3) was received from eBioscience (San Diego, USA). RevertAid First Strand cDNA Synthesis Kit (#K1622) was purchased from Fermentas UAB (Vilnius, Lithuania). FastStart Universal SYBR Green Master (Rox) (04913914001) was purchased from Roche (Mannheim, Germany). Endothelial cell medium and HPMECs were purchased from ScienCell (Carlsbad, USA).

Ovcar3 and Ovcar8 cells subjected to scrambled or Caspase 8 shRNA knockdown were first selected with puromycin as described before^{4 (link)}. After selection, cells were transduced with a lentiviral vector containing an NFκB transcriptional regulatory element, using the Cignal Lenti Reporter System (CLS-013L), according to the manufacturer’s specifications and allowed 72 h for maximum vector expression. Transient reporter assays were subsequently performed for 18 h. Briefly, cells were plated in 96-well plates at a density of 10,000 cells/well. After overnight attachment, cells were exposed to serum starvation medium containing 0.5% fetal bovine serum for 24 h. Drugs or IKKβ inhibitor IV (EMD Biosciences) were added for 1 h after which TNFα (300-01A, PeproTech) was added to stimulate NFκB activity for 18 h. Control wells received vehicle alone. Luciferase activity was measured using the Luciferase Assay System (E4030, Promega) according to the manufacturer’s instructions and a Spectramax M5 plate reader (Molecular Devices). Luciferase units were normalized to viable cell number, obtained by XTT assay, on duplicate assay plates. Experiments included 3–6 replicate samples per point and were repeated at least three times.

LPS (L6529), puromycin (P8833), and PMA (P8139) were purchased from Sigma-Aldrich. TNF-α (300-01A) and IL-1β (200-01B) were from Peprotech. Cell lysis buffer (9803) was from Cell Signaling. Phosphatase Inhibitor Cocktail 3 (P0044) and Protease inhibitor cocktail (P8340) were from Sigma-Aldrich. Dynabeads Protein G (10004D), Dynabeads Protein A (10001D), SuperSignal West Femto Maximum Sensitivity Substrate (34096), and Pierce BCA Protein Assay (23225) were from Thermo Fisher Scientific. Dual-Luciferase Reporter Assay System (E1910) was from Promega. Nitrocellulose membrane (1620115) was from Bio-Rad. Polyethyleneimine (PEI) (24313-2) was purchased from Polysciences. Purified recombinant IKKα (TP761707) and IKKβ (TP750220) were purchased from Origene.

TNFα (210-TA) (for in vivo experiments) was from R&D system; D-GalN (101743) was from Millipore; ConA, CHX, puromycin and doxycycline (DOX) were from Sigma Aldrich; ZVAD-FMK (s7023) and sp600125 (s1406) were from Selleck; LCL161 (HY-15518) was from MCE; TNFα (300-01 A) (for in vitro experiments) was from Peprotech; Flag-TNF was from Enzo Life (ALX-522-009-C050); Lipofectamine 2000 Transfection Reagent was from Invitrogen. The following antibodies were used for flow cytometry: FITC-Annexin V (556420), 7-AAD (559925) were from BD Biosciences; Alexa Flour^® 488 anti-rabbit IgG was from Invitrogen. The following antibodies were used for immunoprecipitation and immunoblotting: anti-Bcl-3 (sc-185), anti-Caspase 8 (sc-6136), anti-STAT3 (sc-482), anti-UB (sc-8017), anti-rabbit IgG (sc-2027) and anti-mouse IgG (sc-2025) were from Santa Cruz; anti-Caspase 8 (9746), anti-cleaved Caspase 3 (9661), anti-pJNK (4668), anti-JNK (9252), anti-pIκB (2859), anti-PARP (9542), anti-FADD (2782), anti-A20 (5630), and anti-CYLD (8462) were from Cell Signaling Technology; anti-HA tag (51064) and anti-MYC tag (60003) were from Proteintech; anti-cIAP1/2 (MAB3400) was from R&D; anti-pSTAT3 (CY6566) was from Abways; anti-RIP1 (610458) was from BD Biosciences; anti-β-Actin (A2228), anti-Flag (F3165) and anti-Flag M2 affinity gel (A2220) were from Sigma Aldrich; anti-GAPDH (KC-5G4).

Monoclonal rabbit anti-NLRP3 (D2P5E, 13158 S), anti-caspase-9 (9502 T), anti-caspase-8 (1C12, 9746 S), anti-caspase-3 (9665 S), anti-cleaved-caspase-3 (Asp175, 9661S), anti-BID (2002S), anti-caspase-1 (2225S), anti-MLKL (14993S), Anti-pMLKL (S358, used in western blotting, 91689S), and anti-PARP-1 (9542S) were purchased from Cell Signaling Technology (Beverly, MA, USA). Polyclonal antibody anti-IL-1β (A1112) was acquired from Abclonal (Wuhan, China). Polyclonal rabbit anti-α-tubulin (11224-1-AP) was purchased from Proteintech (Chicago, IL, USA). Anti-pMLKL (S358, used in immunofluorescence assay and IHC, ab187091) was purchased from Abcam (Cambridge, UK). Anti-pMLKL (S345, used in IHC, MA5-32752) was purchased from Thermo Fisher (Waltham, MA, USA). Rabbit anti-RP3-NP antibody (RP3) and mouse-anti-SARS-CoV-2-NP (SR24) were made in-house.
LPS (L4391) was obtained from Sigma-Aldrich (St. Louis, MO, USA). VX-765 (S222), MCC950 (S7809), Z-VAD-FMK (S7023), Z-IETD-FMK (S7314), Z-DEVD-FMK (S7312), and NSA (S8251) were obtained from Selleck (Houston, TX, USA). MLN120B (HY-15473), GSK-872 (HY-101872), SM-164 (HY-15989), and staurosporine (HY-15141) were purchased from MedChemExpress (New Jersey, USA). TNF-α (300-01A) was obtained from Peprotech (Rocky Hill, NJ, USA). DAPI (4,6-diamidino-2-phenylindole, C1002) was purchased from Beyotime (Shanghai, China).