P eif2α

Eicosapentaenoic Acid (purity, ≥97%), 3-Methyladenine (3-MA, a PI3K inhibitor) and Dorsomorphin (Compound C, an AMPK inhibitor) were purchased from MedChemExpress (NJ, United States). Type II collagenase, TBHP, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, United States). Primary antibodies for cleaved-caspase 3 (C-C3), Beclin-1, LC3B, p-mTORC1, mTORC1, BCL-2, AMPK and p-AMPK were procured from Cell Signaling Technologies (Danvers, MA, United States). Antibodies against BAX, BCL-2, Collagen II, MMP13 and P62 were purchased from Abcam (Cambridge, United Kindom). Antibodies against p-PERK, PERK, p-eIF2α, eIF2α, CHOP, ADAMTS5, Aggrecan, and ATG5 were purchased from ABclonal (WH, CHINA). Antibodies against ATF4, GRP78, Collagen II, and β-actin were purchased from Proteintech (NJ, China). Horseradish peroxidase-labeled secondary antibodies, Alexa Fluor^® 488-labeled goat anti-rabbit IgG (H + L) secondary antibody, and Alexa Fluor^® 594-labeled goat anti-mouse IgG (H + L) secondary antibody were purchased from Abcam. Further, 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (SH, CHINA). The reagents for cell culture were obtained from Gibco (Grand Island, NY, United States).

Cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) and phosphate protease inhibitor. Protein samples were harvested, boiled, separated with 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% milk and incubated overnight at 4°C with primary antibodies against GAPDH (1 : 2000, ZSGBBIO, Beijing, China), USP7 (1 : 1000, HUABIO, Hangzhou, China), collagen type II alpha 1 chain (Col2a1, 1 : 1000, HUABIO, Hangzhou, China), sex-determining region Y-box 9 (Sox9, 1 : 2000, Abcam, Cambridge, UK), Cleaved Caspase-3 (1 : 1000, CST, MA, USA), Bcl-2 (1 : 1000, ABclonal, Wuhan, China), Bcl-2-associated X (Bax, 1 : 1000, ABclonal, Wuhan, China), eIF2α (1 : 1000, ABclonal, Wuhan, China), eIF2α phosphorylation (p-eIF2α, 1 : 1000, ABclonal, Wuhan, China), activating transcription factor 4 (ATF4, 1 : 1000, HUABIO, Hangzhou, China), CHOP (1 : 300, Santa Cruz, CA, USA), p65 (1 : 1000, CST, MA, USA), p65 phosphorylation (p-p65, 1 : 1000, CST, MA, USA), and proliferating cell nuclear antigen (PCNA, 1 : 1000, HUABIO, Hangzhou, China). Then, they were incubated in horseradish peroxidase-conjugated secondary antibodies (1 : 10000, Biosharp, Hefei, China) and visualized with an electrochemiluminescence reagent.

For protein extraction, cells were lysed with RIPA buffer (Biosharp, Hefei, China) containing protease inhibitors (MCE). Proteins were quantified with a BCA assay kit (Vazyme) and western blotting was done using standard procedures, as previously reported [41 (link)]. Polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany) were utilized to transfer proteins. We detected signals and acquired images using Electrochemiluminescence (ECL) luminescent solution (Biosharp) and ChemiDoc XRS+ imaging system (BioRad). Antibodies against PCNA (Servicebio, GB11010), E-cadherin (Abclonal, A20798; Wuhan; China), N-cadherin (Abclonal, A19083), BiP (Cell Signaling Technology, 3177 S; Danvers; MA; USA), eIF2α (Cell Signaling Technology, 2103 S), p-eIF2α (Abclonal, AP0692), CHOP (Proteintech, 15204-1-AP; Wuhan; China), GDF15 (Abclonal, A0185), PPIB (Abcam, ab178397; Cambridge; MA; USA), Bax (Abclonal, A0207), Bcl-2 (Abclonal, A0208), β-actin (Cell Signaling Technology, 8457 S) were used in our study. Original Western blots are also shown as supplementary information.