P perk

Curcumin, tert-Butyl hydroperoxide solution (TBHP), thapsigargin (TG), DAPI, type II collagenase, and EX527 were purchased from Sigma-Aldrich (St. Louis, MO, USA). 0.25% trypsin was purchased from Gibco (NY, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium- (DMEM-) F12 medium and PBS were obtained from HyClone (Logan, UT, USA). TUNEL staining kit was purchased from Beyotime (Jiangsu, China). The primary antibodies against SIRT1, cleaved caspase3, cleaved PARP, ATF4, GRP78, CHOP, PERK, p-PERK, eIF2α, p-eIF2α, and β-actin were purchased from Cell Signaling (Danvers, MA, USA); Bcl2 antibody was purchased from Abcam (Cambridge, UK). Cell counting kit-8 (CCK-8) was obtained from DOJINDO (Kumamoto, Japan).

The expression of five important proteins involved in the PERK-eIF2α-ATF2 pathway was examined in a western blot assay. The primary antibodies were against PERK (1:1,000; #3192; Cell Signaling Technology, Inc., CST), P-PERK (1:1,000; #3179; Cell Signaling Technology, Inc.), eIF2α (1:1,000; #2103; Cell Signaling Technology, Inc.), P-eIF2α (1:1,000; #9721; Cell Signaling Technology, Inc.) and ATF2 (1:1,000; #9226; Cell Signaling Technology, Inc.). The secondary antibody was horseradish peroxidase (HRP)-labeled anti-rabbit and mouse IgG (H+L), which is polyvalent. The internal reference was a GADPH antibody (1:5,000; KC-5G5; Kandchen Bio-tech Inc., Shanghai, China). The whole procedure was based on a previous protocol (16 (link)). Based on the intensity of the protein bands, IOD was calculated using Gel-Pro Analyzer 4 software (Media Cybernetics, Inc.) for statistical study. Data was analyzed using the SPSS 17.0 statistics software package. Quantitative data were presented as the mean ± SD, and qualitative data as a percentage. One-way analysis of variance (ANOVA) was applied to make comparisons within one group and ANOVA was used to make mean comparisons in groups. A two-sided test was used to check statistics.

The antibodies for histone H3 and acetyl-histone H3, α-tubulin and acetyl-α-tubulin, p53 and p21 were obtained from Abcam (Cambridge, UK). Apaf-1, CDK 4, CDK 6, cyclin D1, Bcl-2, p-NFκB, caspase-3, caspase-9 and β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for GRP78, ATF4, CHOP, PERK, p-PERK, eIF2α and p-eIF2α were purchased from Cell Signaling Technology (Danvers, MA, USA). The Bax antibody was obtained from Novus Biologicals (Littleton, CO, USA).

Liver tissues were homogenized and determined as described previously (Wang et al., 2019 (link)). The blots were incubated with primary antibodies: cleaved caspase-3, Bax, Bcl-2, ATF6, GRP78, Nrf2, SOD2, collagen IA1, ATG5, p62 and LC3 (Abcam, USA), HO-1 (Santa Cruz Biotechnology, CA, USA), CHOP, P-IRE1α, P-eIF2α, P-PERK, PERK, FGFR1, P-AMPK and AMPK (Cell Signaling Technology, Danvers, MA, US), and α-SMA, TGF-β, and GAPDH (Proteintech, China) followed by incubation with their corresponding secondary antibodies: anti-rabbit and anti-mouse (Proteintech, China).

Quercetin (CAS NO: 117-39-5, purity > 95%, Figure 1A) was purchased from Sigma-Aldrich. Kits for detecting SOD and MDA were purchased from Nanjing Jiancheng Bioengineering Institute. The antibodies for PSD93, PSD95, SIRT1, Caspase3, Bax, Bcl2, PERK, P-PERK, eIF2α, P-eIF2α, IRE-1α, P-IRE-1α, BIP, PDI were provided by Cell Signaling Technology (MA, USA). Anti-β-actin, BDNF, NGF, ATF6 were purchased from Abcam, Inc (Cambridge, England).

We lysed the cells in RIPA buffer (Sigma Aldrich) containing 150 mM NaCl, 1.0% Nonidet-P 40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tri (pH 8.0), protease inhibitor cocktail, and PhoSTOP (Roche Molecular Biochemicals, Basel, Switzerland) as described previously^{17 (link)}. We used the following primary antibodies: PPARγ, C/EBPα, CHOP, GRP78, PERK, p-PERK, p-eIF2α, eIF2α, IRE1α, IRE1α, FABP4, perilipin, and α-tubulin (1:1000; Cell Signaling Technology, Danvers, MA, USA).
Secondary antibodies (anti-rabbit IgG or anti-mouse IgG, 1:2000)) were purchased from Cell Signaling Technology. We conducted immunoreactive detection of specific proteins using an ECL Western blotting kit (GE, Hercules, CA, USA) according to the manufacturer’s instructions.