Z vad fmk

The Ishikawa, HEK293T and H1299 cell lines were cultured in DMEM (Corning, 10-013-CVR) supplemented with 10% (v/v) FBS (Gibco, 10099141). The HEC-265 cell line was cultured in EMEM (M&C Gene Technology, CM10010) with 15% FBS. The AN3CA cell line was cultured in EMEM supplemented with 10% FBS, 1× NEAA (Gibco, 11140050) and 1 mM sodium pyruvate (M&C Gene Technology, CC007). The cell lines were originally purchased from ATCC or Cell Resource Center of IBMS-CAMS, freshly thawed from our stock and cultured for no longer than 2 months. All cell lines were negative for mycoplasma contamination. The transfections were conducted by using Lipofectamine 2000 (Invitrogen, 11668500) according to the manufacturer’s protocol. To generate a luciferase reporter, annealed oligos were cloned into a pGL3-basic vector (Promega, E1761). MPA was purchased from Selleck (S2567). The following reagents were used as: Z-VAD-FMK (Solarbio, IZ0050) 10 µg/ml; necrostatin-1 (Sigma‒Aldrich, N9037) 10 µg/ml; ferrostatin-1 (Sigma‒Aldrich, SML0583) 2 µM; 3-MA (Sigma‒Aldrich, M9281) 2 mM; PD146176 (Selleck, S6956) 5 µM; liproxstatin-1 (Selleck, S7699) 2 µM; UAMC-3203 (Selleck, S8792) 2 µM. The antibodies and the sequence of siRNA, sgRNA and primers are listed in Supplementary Table S1.

Porcine kidney-15 (PK-15), human embryonic kidney (HEK293T) cells, and baby hamster kidney cells (BHK-21) cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained at 37°C with 5% CO₂. The SVA virus (GenBank accession number: MZ375462) was used for all the experiments. The proteasome inhibitor MG-132, the lysosomal inhibitor chloroquine (CQ), and the caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoro-methyl ketone (Z-VAD-FMK) was purchased from Beijing Solarbio Science & Technology Co. Ltd.

Reagents used are as follows: erastin (#E7781, Sigma Aldrich), ferrostatin-1 (Fer-1) (#RM02804, ABclonal), liproxstatin-1 (Lip-1) (#RM02805, ABclonal), desferrioxamine mesylate (DFO) (#RM02807, ABclonal), necrostatin-1 (#RM02815, ABclonal), chloroquine (#RM02814, ABclonal), and Z-VAD-FMK (#IZ0050, solarbio).
Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) (LJ621, Dojindo, Japan). 5000 cells per well were seeded in a 96-well plate; upon a 24-hour treatment, each well was replaced with fresh medium containing CCK-8 reagent. After incubation for 2 h at 37°C, then, cell viability was measured on a microplate reader (Synergy HT, Bio-Tek, USA) at 450 nm.