Z vad fmk zvad

The HT29 human colorectal carcinoma cell line was purchased from Dainihon Pharmacy Co. (Tokyo, Japan). TMK-1 human gastric carcinoma cells were a gift from Professor Wataru Yasui (Molecular Pathology, Hiroshima University, Hiroshima, Japan), and the CT26 murine colon carcinoma cell line was a gift from Professor I. J. Fidler (MD Anderson Cancer Center, Houston, TX, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO₂.
BBR (25 μM for 48 h unless otherwise specified, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), 5-fluorouracil (5-FU), N-acetyl-L-cysteine (NAC 1 mM), rotenone (0.5 μM) (Sigma-Aldrich Inc., St. Louis, MO, USA), malonate (0.5 mM), vitamin E (20 μM) and vitamin C (1000 μM) (Fujifilm-WAKO Chemicals, Osaka, Japan), Z-VAD-FMK (ZVAD, 20 μM) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ferrostatin-1 (FRS, 2 μM), deferoxamine (DFO, 200 μM) (Cayman Chemicals, Ann Arbor, MI, USA), were purchased from the manufacturers listed.
Cell death was detected using Live-or-Dye™ Fixable Viability staining kit (Cosmobio, Tokyo, Japan). Dead cells were counted by observation of 500 cells. Apoptotic figures were detected by Hoechst 33342 dye (Sigma) apoptotic figures were counted by observation of 500 cells.

Human embryonic kidney cells (HEK293T, cat #SCSP-502), human lung alveolar epithelial cells (A549, cat #TCHu150) and wild type respiratory syncytial virus (RSV) long strain (Cat #GDV052) were purchased from Cell Bank of Chinese Academy of Sciences (CCTCC). Recombinant Adenovirus (ADV) that is packaged in HEK293T cells with the plasmid pSG500-GFP and pBGHlox and that could be replicated in tumor cells was provided by Sinogenomax Inc., Beijing, China. Primary antibody against forphospho-IRF-3 (S386) was purchased from Abcam. Primary antibodies for caspase-9 and -3 were purchased from Cell Signaling Technology. Primary antibodies for 6×His-tag, RSV fusion protein, RSV glycoprotein G, RIP1, RIP3 and the mixed lineage kinase domain like protein (MLKL), and horseradish peroxidase or fluorescent labeled secondary antibodies of goat anti-mouse IgG, goat anti-rabbit IgG and rabbit anti-goat IgG were purchased from Santa Cruz Biotechnology. Caspase inhibitor zVAD-fmk (zVAD), necroptosis blocking agent necrostatin-1-fmk (Nec-1) and cathepsin D inhibitor pepstatin A (PepA) were purchased from Santa Cruz Biotechnology, TNF-α and Bovine serum albumin (BSA) from Sigma-Aldrich, and propidium iodide (PI) from Beyotime Institute of Biotechnology, China.

For the cell death inhibitor assay, H9c2 cells were treated with human recombinant HMGB1 (40 μg/mL, Biolegend, San Diego, CA, USA) for 48 h, with or without the addition of cell death inhibitors. The following inhibitors were utilized: N-acetyl-L-cysteine (NAC, 1 mM, Sigma, St. Louis, MO, USA), Z-VAD-FMK (ZVAD, 20 μM, Santa Cruz Biotechnology, Santa Cruz, CA, USA), ferrostatin-1 (FRS, 2 μM), deferoxamine (DFO, 200 μM, Cayman Chemicals, Ann Arbor, MI, USA), and 4-phenylbutyric acid (4PBA, 200 μM, WAKO). Additionally, chloroquine (100 µM, WAKO) was employed to inhibit autophagy.