Z vad fmk

The small interfering RNA against circ_0008035 (si-circ_0008035), mimics of miR-599 (miR-599), inhibitors of miR-599 (anti-miR-599), the overexpression vector of EIF4A1 (EIF4A1), short hairpin RNA targeting circ_0008035 (sh-circ_0008035) and their corresponding controls (si-NC, miR-NC, anti-miR-NC, vector and sh-NC) were synthesized by RIBOBIO (Guangzhou, China). The oligonucleotides or plasmids were transiently transfected into GC cells using Lipofectamine 2000 (Invitrogen).
GC cells were treated with ferroptosis inducer erastin (10.0 μM; Solarbio, Beijing, China), ras selective lethal 3 (RSL3; 2.0 μM; Sigma, St. Louis, MO, USA), erastin (Solarbio) + ferroptosis inhibitor ferrostain-1 (2.0 Μm; Abcam, Cambridge, MA, USA), erastin (Solarbio) + apoptosis inhibitor ZVAD-FMK (10.0 μM; Sigma), erastin (10.0 μM; Solarbio) + necroptosis inhibitor necrosulfonamide (0.5 μM; Abcam), RSL3 (2.0 μM; Sigma) + ferrostain-1 (Abcam), RSL3 (2.0 μM; Sigma) + ZVAD-FMK (10.0 μM; Sigma) or RSL3 (2.0 μM; Sigma) + necrosulfonamide (0.5 μM; Abcam) for 48 h for the subsequent experiments.

Honokiol was purchased from Wako (Osaka, Japan). 3-(4,5-dimethylthiazol-2-yl)−2, 5-Diphenyltetrazolium bromide (MTT), NAC, PD98059, and dimethyl sulfoxide (DMSO) were obtained from Sigma (St. Louis, MO, USA). MEM medium, fetal bovine serum (FBS), penicillin, streptomycin, and phosphate-buffered saline (PBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Primary antibodies, including cleaved-poly (ADP-ribose) polymerase (PARP), Atg7, LC3B, p-ERK, ERK and GRP78, together with GAPDH antibodies and secondary antibodies, were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The broad-spectrum caspase inhibitor (z-VAD-fmk) was obtained from Millipore (Billerica, MA, USA). 3-MA was purchased from Selleckchem (Houston, TX, USA). Antibodies against caspase-3, caspase-9, Bcl-2, Bcl-xl, survivin, Cyclin D1, Cyclin E, and Cdk4 were purchased from Abcam.

ORM was procured from HLL Lifecare Limited. JC-1, antibiotic, AlexaFluor 488 and 568 conjugated antibodies, Lipofectamine 2000 were purchased from Invitrogen; z-VAD-fmk, protease inhibitor cocktail and GAPDH antibody were from Millipore. FITC-conjugated Annexin V was from BD Pharmingen. Beclin 1 and scrambled siRNA were from Dharmacon. Antibodies for cleaved PARP, LC3, Caspase 3, IRE1α, PERK, GRP78/BiP, p-p70 S6k, mTOR, p-mTOR, p-Akt as well as LC3 siRNA were purchased from Cell Signaling Technology. Beclin 1, Atg5 and β-actin antibodies were from Sigma. Antibodies for ATF6, CHOP/GADD153 and all secondary antibodies were from Santa Cruz. Spurr embedding kit was from Ted Pella. All other chemicals were from Sigma.

We obtained an In Situ Cell Death Detection Kit (Fluorescein), thiazolyl blue tetrazolium bromide (MTT), indomethacin, quercetin, Dulbecco’s modified Eagle’s medium (DMEM), DNase, and other reagents and chemicals from Sigma–Aldrich, Merck KGaA (Darmstadt, Germany). We obtained Muse Caspase-3/9 Assay Kits and the pan-caspase inhibitor Z-VAD-FMK from Millipore, Merck KGaA (Darmstadt, Germany). We obtained dihydroethidium, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA), 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)], and MitoSOX from Molecular Probes, Thermo Fisher Scientific (Waltham, MA, USA). We purchased L-glutamine, fetal bovine serum (FBS), trypsin-EDTA, and penicillin/streptomycin from HyClone, GE Healthcare Life Sciences (Logan, UT, USA).

The following reagents were obtained commercially: rabbit anti-caspase-3 from Cell Signaling Technology (Danvers, MA, USA); rabbit anti-HMGB1 from Abcam (Cambridge, UK); mouse anti-β-actin, H₂O₂, t-BHP, propidium iodide (PI), STS, Necrostatin-1 (Nec-1), 3-Aminobenzamide (3AB), 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ), poly-l-lysine, nicotinamide adenine dinucleotide (NAD), SI and 50% glutaraldehyde from Sigma-Aldrich (Saint Louis, MO, USA); horseradish peroxidase (HRP)-conjugated anti-mouse antibody, HRP-conjugated anti-rabbit antibody, fluorescein (FITC)-conjugated anti-mouse antibody and 4′,6-diamidino-2-phenylindole (DAPI) from Thermo Fisher Scientific (Rockford, IL, USA); mouse anti-RIPK1 antibody, mouse anti-PARP-1 antibody, and Annexin V from BD Biosciences (San Jose, CA, USA); rabbit anti-RIPK3 antibody and mouse anti-AIF antibody from Santa Cruz Biotechnology (Dallas, TX, USA); rabbit anti-PAR antibody from Trevigen (Gaithersburg, MD, USA); mouse anti-PAR antibody from Enzo Life Sciences (Farmingdale, NY, USA); z-VAD-fmk from Millipore (Darmstadt, Germany); olaparib from Selleck Chemicals (Houston, TX, USA); UPF-1069 and XAV-939 from Tocris Bioscience (Minneapolis, MN, USA); and MNNG from Tokyo Chemical Industry (Tokyo, Japan).

Human colon cancer cells HT-29 (Cat# 300215) were purchased from CLS (Cell Lines Service, Eppelheim, Germany) and were maintained in DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin (Hyclone, Cramlington, UK). Antibodies against caspase-8 and p27 were obtained from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA); those raised against β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Antibodies raised against Cleaved PARP were obtained from Abcam (Abcam, Cambridge, UK); those raised against p21 and γH2AX were obtained from Millipore (Millipore, Hayward, CA, USA). Chloroquine (CQ) was obtained from Sigma-Aldrich (Sigma-Aldrich, Saint-Quentin Fallavier, France); 3-Methyladenine and Z-VAD-FMK were obtained from Millipore, SB 203,580 was purchased from Cell Signaling and SB 202190 was purchased from Abcam. Antibodies against caspase 8, cleaved caspase 3, mTOR, phospho-mTOR, p70S6, phosphor-p70S6, p38, phosphor-p38, LC3, and Beclin-1 were obtained from Cell Signaling; those against γH2AX were obtained from Millipore, those against TNFα, p62/SQSTMI, and cleaved PARP were obtained from Abcam. The antibody against β-actin was obtained from Santa Cruz Biotechnology.