Ire1α

Recombinant human PDGF was purchased from PeproTech (100-14B, United States). pPERK, ATF6, IRE1α, GRP78, PDI, VPS34, Beclin1, ATG5, LC3II, GAPDH and secondary antibodies were obtained from Abcam (Abcam, United States), PDI was purchased from Cell Signaling Technology (Danvers, MA, United States), NLRP3, Pro-Caspase1, Caspase1, GSDMD, GSDMDP30, IL-1 were obtained from Proteintech (Proteintech, China). Goat anti-rabbit and anti-mouse IgG-HRP were purchased from Cell Signaling Technology (Danvers, MA, United States). An enhanced chemiluminescence (ECL) kit was purchased from Bio-Rad (Hercules, CA, United States). H&E kit was purchased from Beyotime.

Total cellular protein lysates were prepared with Pierce lysis buffer (Pierce, Rockford, IL, USA) and quantified by the Bradford method. Equal amounts of total protein (20 µg) were loaded onto 10% SDS‐PAGE gels and electrophoresed at 100 V for 1 hour. Then, the separated proteins were transferred onto PVDF membranes (Millipore, Billerica, MA, USA) at 80 V for 2 hours. The membranes were blocked in 5% non‐fat milk in 1× TBST and then incubated with primary antibodies against EGFR (1:500; Santa Cruz, USA), PERK, IRE1α, ATF 6, (1:1000; Abcam), phospho‐eIF2α, GRP78, GRP94, PDI, ERO1‐Lα, CHOP, phospho‐ATM, DNA‐PK, LC3B, Atg3, cleaved caspase 3, cleaved PARP, and β‐actin (1:1000; Cell Signaling Technology, Boston, MA, USA) at 4°C overnight. After washing with 1× TBST, the membranes were incubated with secondary anti‐mouse or anti‐rabbit IgG HRP‐linked antibodies (1:5000; Cell Signaling Technology) at room temperature for 2 hours. The blots were developed with Target LumiGLO (Cell Signaling Technology) and photographed with DNR BioImaging System (DNR, Israel).

Total protein extracts were prepared and separated on 12% SDS-PAGE and electrophoretically transferred to nitrocellulose filter membranes. The membranes were blocked with 5% bovine serum albumin. And then the membranes were incubated overnight at 4°C with the indicated primary antibodies. The dilutions of antibodies were prepared as follows: HMGB1 (CST number 3935, 1 : 1000), BSA (Santa Cruz sc-50528, 1 : 1000), PERK (CST number 3192, 1 : 1000), CHOP (CST number 2895, 1 : 1000), IRE1α (CST number 3294, 1 : 1000), ATF6 (Abcam ab62576, 1 : 2000), Bcl₂ (Santa Cruse number KO112, 1 : 500), and β-actin (CST number 5125, 1 : 1000). After washing, membranes were treated with horseradish peroxidase-conjugated secondary antibodies (1 : 2000; CST, number 7074, USA) and then the bands were visualized using the enhanced chemiluminescence kit (Thermo, number 34080, USA).

Cell or liver homogenate proteins of the above groups were extracted, and the concentrations were measured with BCA Protein Assay Kit (Sangon Biotech Co., Ltd., Shanghai, China). Samples were denatured for 10 min at a temperature of 100°C. A total of 40 μg protein was loaded per lane, separated using 12% SDS-PAGE gel, and then electrotransferred onto polyvinylidene difluoride membranes. Membranes were blocked using 5% nonfat milk for 1 h and then incubated at 4°C overnight with primary antibodies against Bad (cat. no. ab32445; 1 : 1000; Abcam Inc.), Bax (cat. no. E63; 1 : 1000; Abcam Inc.), Caspase-3 (cat. no. ab2302; 1 : 1000; Abcam Inc.), Bcl2 (cat. no. ab196495; 1 :1000; Abcam Inc.), p-IRE1α (cat. no. ab48187; 1 : 1000; Abcam Inc.), IRE1α (cat. no. ab37117; 1 : 2000; Abcam Inc.), TRAF2 (cat. no. #4712; 1 : 1000; CST Inc.), p-IκBα (cat. no. #2859; 1 : 1000; CST Inc.), IκBα (cat. no. #9242; 1 : 1000; CST Inc.), p-p65 (cat. no. #3033; 1 : 1000; CST Inc.), p65 (cat. no. sc-71675; 1 : 2000; Santa Cruz Inc.), PPARγ (cat. no. ab209350; 1 : 1000; Abcam Inc.), Arg1 (cat. no. ab60176; 1 :1000; Abcam Inc.), and iNOS (cat. no. ab15323; 1 : 2000; Abcam Inc.). The membranes were blotted with species-matched secondary antibodies. Protein bands were visualized using the BioRad ChemiDoc™ XRS system (Hercules, CA). All images were analyzed using the NIH ImageJ software.

CYN (97% purity) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), and a stock solution (100 mM) in dimethyl sulfoxide (DMSO; ≥99.7%, Sigma‒Aldrich, St. Louis, MO, United States) was stored at −20°C. The final concentrations of DMSO in all experiments were lower than 0.1% (V/V). RAPA (100 nM), 3-Methyladenine (3-MA, 2 mM), chloroquine (CQ, 10 μM) and TUDCA (2 μM) were purchased from MCE (Monmouth Junction, NJ, United States). Triton X-100, 4% paraformaldehyde and a bicinchoninic acid (BCA) protein assay kit were purchased from Beyotime Biotechnology (Shanghai, China). Primary antibodies against Bax, Bcl-2, Beclin-1, LC3B, p62/SQSTM1, Atg5, CHOP, GRP78, ATF6, p-eIF2α, IRE1α and LAMP-2 were purchased from Abcam (Cambridge, United Kingdom). Primary antibodies against PARP, cleaved caspase-3, caspase-3, Ki-67 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States).