Pro caspase 8

Cell lysates and supernatants (reduced and denatured) were separated on 4–12% gradient gels (Invitrogen) and protein transferred nitrocellulose membrane (Amersham) for detection. Membranes were blocked with 5% skimmed milk in PBS containing 0.1% Tween 20 (PBST) for 1–2 h and were then probed overnight with primary antibodies (all diluted 1/1,000 unless noted otherwise) to mouse β-actin (Sigma; A-1978), cIAP1 (1/500 dilution, ALX-803-335;Alexis Bio-chemicals), RIPK3 (Axxora; PSC-2283-c100), RIPK1 (BD Transduction Laboratories; 610458), MLKL (in-house; 3H1), pro and mature IL-1β (R&D Systems; AF-401-NA), pro- and cleaved-caspase-1 (Santa Cruz; sc-514), Adipogen (AG-20B-0042-C100), pro-caspase-8 (in-house), cleaved caspase-8 Asp387 (9429; Cell Signaling) and Ubiquitin (3933; Cell Signaling). Relevant horseradish peroxidase-conjugated secondary antibodies were applied for 1–2 h. Membranes were washed four to six times in PBST between antibody incubations and all antibodies were diluted in PBST containing 5% skimmed milk. Membranes were developed using ECL (Millipore). For images cropped for presentation, the full-size images are presented in Supplementary Figs 9–14.

Minimum essential medium (MEM) and fetal bovine serum (FBS) were purchased from Invitrogen (CA). Antibodies specific to Bcl-2, pro-caspase-3, pro-caspase-8, pro-caspase-9, cleaved-caspase-3, Bax, poly ADP-ribose polymerase (PARP), Bid, truncated Bid (t-Bid), androgen receptor (AR), prostate-specific antigen (PSA), phosphatase and tensin homolog (PTEN), anti-microtubule-associated protein light chain-3 (LC3), and beclin1 were purchased from Cell Signaling Technology Inc. (MA). 3-Methyladenine (3-MA) was obtained from Sigma Aldrich (MO). SLE was extracted with 100% ethanol at room temperature for 24 hours in a shaker, and the filtered extracts were concentrated and powdered under reduced pressure. The powder was lyophilized and stored at 4°C.

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).

Cell lysates together with culture supernatants were collected by adding 5× sample buffer (50% glycerol, 10% SDS, 5% 2-mercaptoethanol, 0.02% bromophenol blue, and 250 mM pH 6.8 Tris-HCl) for immunoblot analysis. Proteins were separated by 10%–15% polyacrylamide gels, followed by electrophoretic transfer to PVDF membranes (IPVH00010, Millipore). The membrane was then blocked by incubation with 5% BSA before being incubated with primary antibodies. Antibodies used include caspase-1 (clone 4B4), a kind gift from Vishva M. Dixit (Genentech, USA), caspase-3 (9662, Cell Signaling), pro-caspase-8 (4790, Cell Signaling), cleaved-caspase-8 (9429, Cell Signaling), caspase-9 (9508, Cell Signaling), GSDMD (ab209845, Abcam), ASC (67824, Cell Signaling), and GAPDH (AC002, ABclonal).

The preparation of SCC-9 and HSC-3 cell lysates for the Western blot analysis followed previously described procedures [18 (link)]. The Western blot analysis was performed with indicated primary and horseradish peroxidase-conjugated secondary antibodies. The primary antibodies were used as follows: pro-caspase-8 (#9746), pro-caspase-9 (#9502), cleaved caspase-8 (#9496), cleaved caspase-9 (#9505), cleaved caspase-3 (#9664), PARP (#9542), p-ERK (#4370), ERK (#9102), p-JNK (#4668), JNK (#9258), cIAP-1 (#7065, Cell Signaling Technology, MA, USA); pro-caspase-3 (610323), p38 (612168), p-p38 (612281, BD biosciences); HO-1 (ab68477), beta-actin (ab8226, Cambridge, MA, USA). Then, HRP-conjugated anti-mouse IgG (5450-0011, Seracare Life Sciences, Milford, MA, USA) or anti-rabbit IgG (5450-0010, Seracare Life Sciences, MA, USA) secondary antibodies were applied. All the blots were carried out with an enhanced chemiluminescence substrate solution (EMD Millipore Corporation, Burling, MA, USA) to produce images. The band intensities were quantified by NIH ImageJ analysis software.