Pro caspase 7

Pevonedistat was synthesized by Millennium Pharmaceuticals, Inc. The following reagents were purchased from their respective companies: recombinant rat TNF-α (Peprotech, Rocky Hill, NJ, USA); caspase inhibitors Z-VAD-FMK and Z-IETD-FMK (R&D Systems, Minneapolis, MN, USA); Necrostatin-1 (Sigma-Aldrich, St. Louis, MO, USA); and Epoxomicin (Sigma-Aldrich). Antisera were purchased from the following companies: β-Actin, cleaved caspase-3, cleaved caspase-8 (p18), cFLIP, cullin-3, IκBα, phospho-IκBα, NEDD8, PARP, pro-caspase-3, pro-caspase-6, pro-caspase-7, pro-caspase-8 (p10), pro-caspase-9 (Cell Signaling, Danvers, MA, USA); MLKL (Millipore, Billerica, MA, USA); CDT1 (Santa Cruz Biotechnology, Dallas, TX, USA); and BID (eBioscience, San Diego, CA, USA). Complete antisera details are provided in Supplementary Information.

The following compounds were used in this study: DATS (purity > 97%; Shenzhen Minn Bolin Biotechnology Co., Ltd., Shenzhen, China), iodoacetamide (IAM; Nanjing Dingguo Changsheng Biotechnology Co., Ltd., Nanjing, China), and mitomycin (Sigma, St Louis, MO, USA). They were dissolved in dimethylsulfoxide (DMSO) for experiments. DMSO at a concentration of 0.02% (w/v) was set as a vehicle control throughout the studies. Analytical grade 30% hydrogen peroxide (H₂O₂; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was diluted with deionized water to indicated concentrations for experiments. The following primary antibodies were used in this study: α-SMA, α1(I) procollagen, and fibronectin (Epitomics, San Francisco, CA, USA); TGF-βRI, TGF-βRII, PDGF-βR, EGF-R, and Bcl-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); cyclin A, cyclin B1, CDK1, CDK2, Bax, pro-caspase-9, cleaved-caspase-9, pro-caspase-8, cleaved-caspase-8, pro-caspase-7, cleaved-caspase-7, pro-caspase-3, cleaved-caspase-3, full-length PARP-1, cleaved-PARP-1, and β-Actin (Cell Signaling Technology, Danvers, MA, USA).

Cells were washed twice with PBS and lysed. The lysate samples were then centrifuged at 14000 × rpm for 20 min at 4 °C. Supernatants were collected, and equal amounts of protein (50 μg per lane) were subjected to electrophoresis on 10% (W/V) SDS-polyacrylamide gels and then transferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk in PBST (PBS and 0.05% Tween 20) for 1 h, washed twice with 0.1% PBST, and incubated with primary antibodies for ATF-4, CHOP, pro-caspase-7, cleaved caspase-7 (Cell Signaling Technologies, MA, USA), Grp-78, pro-caspase-3, cleaved caspase-3, Bcl-2, Bax (Santa Cruz, CA, USA), phospho-AMPK, AMPK, phospho-eukaryotic initiation factor 2α (eIF2α), and eIF-2α (Abcam, Cambridge, MA, USA) for 1 h. The membranes were then washed with 0.1% PBST and incubated with secondary antibodies that were conjugated to horseradish peroxidase for 45 min. Antibody-reactive bands were revealed using enhanced chemiluminescence reagents (Amersham Biosciences, Sweden) and exposed to Fuji radiographic film. The protein expression of β-actin was as an internal control. The protein expressions were quantified by densitometry and analyzed by ImageQant TL 7.0 software.