GYY4137 (SML0100; chemical structure shown in Fig. 1A), tert-Butyl hydroperoxide solution (416665), N-acetylcysteine (NAC, A9165), chloroquine (CQ, C6628) were obtained from Sigma-Aldrich; Merck KGaA. DMEM/F12 (SH30023.01), FBS and type II collagenases were purchased from HyClone; Cytiva. 2,7-Dichlorofluorescein diacetate (DCFH-DA) was purchased from Beyotime Institute of Biotechnology. Lipofectamine 2000, calcium green-5N and ER-tracker were both obtained from Invitrogen; Thermo Fisher Scientific, Inc. Primary antibodies [GRP78, #3177; ATF6, #65880; CHOP, #2895; phospho-p70S6 kinase, #9208; p70S6 kinase, #2708; mammalian target of rapamycin (mTOR), #2983; phospho-mTOR, #5536; SQSTM1/p62, #23214; LC3A/B, #4108] were provided by Cell Signaling Technology, Inc. and secondary antibodies (#SA00001-1 and #SA00001-2) were purchased from ProteinTech Group, Inc., respectively.

GYY4137 (100 μM) reduces CH cell numbers without inducing apoptosis. (A) Chemical structure of GYY4137. (B) CH metabolic activity was analyzed by performing an MTT following treatment with GYY4137 dose range (EC50, 112.5±2.5 μM; n=4). (C) Quantification of CHs viability following treatment with different time with GYY4137 treatment. (D) Quantification of CHs viability following treatment with increasing concentrations of TBHP (E) Quantification of CHs viability following treatment with different time with 20 μM TBHP. (F) Quantification of CH viability following different dose of GYY4137 treatment following co-incubation with or without TBHP 20 μM. Data are presented as the means ± SEM (n=3). (C-F) *P<0.05 and **P<0.01 vs. control. CH, chondrocyte; TBHP, tert-Butyl hydroperoxide.

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