Cleaved caspase 8

B-MD-C1 (wt) was purchased from ATCC, DOX (Adriamycin, ADR) was purchased from Hisun pharmaceuticals, Zhejiang, China and dissolved in sterile saline solution (2 mg/ml) and stored at -20 °C before use. Fetal bovine serum (FBS) and RPMI 1640 medium were purchased from Gibco, USA; Lipofectamine™2000 was purchased from Life Technologies, USA; antibodies against CRT, ERp57, caspase-8 and cleaved-caspase-8 were purchased from Abcam, USA; antibodies against p-PERK, eIF2α and p-eIF2α were purchased from Cell Signaling Technology, USA; β-actin and horseradish peroxidase-labeled antibodies were obtained from Santa Cruz, USA. MTT assay kit was obtained from Amresco, USA.

ANXA1 (Ac2-26) (acetyl-AMVSEFLKQAWFIEEQEYVQTVK) was purchased from Hangzhou Angtai Biotechnology Co. Ltd. (China). The Cre and BUN ELISA kits were obtained from Nanjing Jiancheng Bioengineering Institute (China). The p-PI3K, PI3K, p-AKT, AKT, NF-κB, cleaved caspase-3, cleaved caspase-8 and FADD primary antibodies were obtained from Abcam (USA). The Bcl-2 and Bax primary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The TUNEL kit was an In Situ Cell Death Detection Kit and was purchased from Roche (USA). The CCK-8 kit was obtained from Dojindo Molecular Technologies (Gaithersburg, MD, USA). The Annexin V-FITC flow cytometry apoptosis kit was purchased from Tianjin Sungene Biotech Co., Ltd. (China).

We gently rinsed the cells in the six-well plate with phosphate-buffered saline (PBS) and added 200–300 μL of preconfigured radioimmunoprecipitation assay buffer and phenylmethylsulfonyl fluoride (99 : 1) cell lysate to each well. After completing cell lysis, the sample was centrifuged at 4°C (12,000 × g, 10 min). Then, we added 5× loading buffer (1/5^th of the supernatant volume) and placed the tube containing this mixture in a 100°C water bath for 10 min. After 10% SDS-PAGE, the bands were transferred to a polyvinylidene fluoride membrane and incubated with specific antibodies against ANGPTL4 (1 : 1000), NOD-like receptor thermal protein domain associated protein 3 (NLRP3, 1 : 2000), B-cell lymphoma 2 (BCL-2, 1 : 2000), apoptosis-associatedspeck-like protein containing CARD (ASC, 1 : 1000), cellular FLICE-like inhibitory protein (cFLIPL, 1 : 2000), caspase-8 (1 : 1500), cleaved caspase-8 (1 : 1500), and β-actin (1 : 2000) (Abcam, Cambridge, MA, USA). The next day, after incubation with IgG (H&L)-HRP antibody (1 : 5000), the bands were visualised on an integrated chemiluminescence imager helped by an enhanced chemiluminescence exposure solution.