Disulfiram

Erastin (S7242), Z-VAD-FMK (S7023), 3-methyladenine (S2767) and disulfiram (S1680) were obtained from Selleck. Ferrostatin-1 (SML0583-5MG) was obtained from Merck.

Copper chelators elesclomol (S1052), clioquinol (S4601), TETA 2HCl (S6585) and disulfiram (S1680) were purchased from Selleck (Houston, TX, USA). Lipofectamine 3000 (L3000015) was obtained from Thermo Fisher Scientific (Manassas, VA, USA). Antibodies for β‐actin (#3700), HA‐Tag (#2367), Flag‐Tag (#14793), SLC7A11 (#12691), CD44 (#37259), Ubiquitin (#3936), OTUB1 (#3783) and Ki67 (#9449) were obtained from Cell Signaling Technology. Antibodies for ATP7A (ab131400) and ATP7B (ab131208) were purchased from Abcam (Cambridge, UK).

Antibodies used for immunoblotting in this study were: rabbit anti-GSDMD antibody EPR19829 (Abcam, Cambridge, UK), rabbit anti-DFNA5/GSDME antibody EPR19859 (Abcam,), rabbit anti-γHexokinase #4959–9988 (Bio-Rad, Hercules, CA, USA), mouse anti-FLAG T0003 (Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-caspase-1 (A-19) (Santa Cruz Biotechnology, Dallas, TX, USA), rat anti-caspase-2 #MAB3507 (Merck, Darmstadt, Germany), mouse anti-caspase-3 (clone 19) (BD BioSciences, Franklin Lakes, NJ, USA), rabbit anti-caspase-4 #4450S (Cell Signaling Technology, Danvers, MA, USA), mouse anti-caspase-5 (4F7) MBL International, Woburn, MA, USA), rabbit anti-caspase-6 (ARC0031 (Thermo Fisher Scientific, Waltham, MA USA), rabbit anti-caspase-7 (#9492) (Cell Signaling Technology), mouse anti-caspase-8 (1C12) (Cell Signaling Technology), rabbit anti-caspase-9 (#9502) (Cell Signaling Technology), rabbit anti-mouse IgG-HRP #A9044 (Sigma Aldrich, Burlington, MA, United States), donkey anti-rabbit-HRP #NA934 (Amersham, Little Chalfont, UK).
Q-VD-OPh was purchased from SMBiochemicals (Anaheim, CA, USA). Disulfiram was purchased from Selleckchem (Houston, TX, USA). TC13172 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). All drugs were dissolved in dimethyl sulfoxide (DMSO) at 10 mM. The final concentration of the DMSO solvent in the assays was 1% regardless of drug concentration.

The renal fibroblasts and the renal epithelial cell lines HK-2 and A498 cells were stimulated with CFT073 wild-type and mutants at a multiplicity of infection (MOI) of 1 or 10 for 6 h and incubated at 37 °C with 5% CO₂. The fibroblasts were also pre-incubated with DMSO (vehicle), p38 MAPK inhibitor SB203580 (10 µM, Santa Cruz Biotechnology Inc., Heidelberg, Germany), ERK1/2 inhibitor PD98059 (10 µM, Santa Cruz Biotechnology Inc.), NF-κB inhibitor BAY 11-7082 (5 µM, Enzo Life Sciences, Farmingdale, NY, USA) and the PKC inhibitor bisindolylmaleimide I (10 µM, Santa Cruz Biotechnology Inc.), JNK inhibitor SP600125 (10 μM, InSolutionT M JNK Inhibitor II, Calbiochem, San Diego, CA, USA), serine protease inhibitor 3,4-Dichloroisocoumarin (DCI, 100 μM, Merck Millipore, MA, USA), cathepsin B inhibitor CA074 (100 μM, Apexbio Technology LLC, Houston, TX, USA), NLRP3 inhibitor MCC950 (2 μM, Avistron Chemistry Services, Cornwall, UK), Disulfiram (10 μM, Selleckchem, Houston, TX, USA), caspase-3 inhibitor AC-DEVD-CHO (10 μM, Enzo Life Sciences) for 1 h prior to CFT073 stimulation for 4 or 6 h at MOI 1. Supernatants and total RNA were collected and kept at −80 °C until further analysis.

Human umbilical vein endothelial cell (HUVECs) and Human aortic smooth muscle cell (HASMCs) were purchased from AmericanType Culture Collection. HUVECs were cultured in EC growth medium bullet kit (EGM-2, Lonza). HASMCs were grown in smooth muscle cell growth medium-2 (SmGM-2, Lonza). Both cells were not used beyond passage 15. H₂O₂ (Sinopharm Chemical Reagent Co., Ltd) was used to induce endothelial cell senescence at a concentration of 100μM for 8 h. Palmitate acid (Sigma-Aldrich) was used to induce cell senescence at a concentration of 200μM for 16 h. To inhibit the role of H₂O₂ or Palmitate acid, HUVECs were pretreated with 5 mM glycine (Sigma-Aldrich) for 4 h or 50uM disulfiram (Selleck) for 1h before stimulation with H₂O₂ or Palmitate acid, and cell lysates and precipitated supernatants were then subjected to the subsequent experiments. LDH assays were performed using LDH-Glo™ Cytotoxicity Assay (Promega) according to the manufacturer’s instructions.

ABT-263, AZD5991, Mitoxantrone, PF-543, Thapsigargin, KIRA6, GSK2606414, Necrostatin-1, Disulfiram and z-IETD-fmk were all purchased from Selleckchem (Houston, TX, USA). Fumonisin B1 was from Cayman Chemicals (Ann Arbor, MI, USA). All compounds were prepared in a DMSO vehicle. Anti-Calreticulin, anti-phospho eIF2α Ser51, anti-full length Caspase 8, anti-Bap31, anti-Cleaved PARP, anti-GRP78/BiP, anti-Bcl-XL, anti-FLIP, anti-full length BID, and anti-total SphK1 antibodies were from Cell Signaling Technologies (Beverly, MA, USA). Anti-GAPDH, and anti-CerS6 antibodies were from Santa Cruz Biotechnologies (Dallas, TX, USA). Anti-phospho-SphK1 Ser225 antibodies were from ECM Biosciences (Versailles, KY, USA). Anti-CerS5 antibodies were from LSBio (Seattle, WA, USA).