Beclin 1

mPEG-poly lactic-co-glycolic acid (PLGA) (75:25, 45,000) was purchased from Daigang Biomaterial Co., Ltd (Jinan, Shandong, China). EGCG (purity > 98%; Yuanye Biotechnology Co., Ltd, Shanghai, China) was dissolved in water (pH 3.0) and stored at −20°C. Nimodipine was obtained from Ruiyang Pharmaceutical Co., Ltd (Yi Yuan, Shandong, China). Lactate dehydrogenase (LDH), glutathione (GSH), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) kits were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). JC-1, Fluo-3 AM, DCFH-DA, Nissl staining kits, and enhanced chemiluminescence kits were purchased from Beyotime (Shanghai, China). CaMKII, Atg5, Beclin-1, and Mn-SOD antibodies were obtained from Proteintech Group, Inc (Rosemont, IL, USA). SP-9002 SPlink detection and DAB kits were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China).

Baicalein (≥ 99%, Yousi Scientific Co., Ltd, Shanghai, China) was dissolved in DMSO at concentration of 200 mM and stored at -20 ℃. Mdivi-1, an inhibitor of Drp1, was purchased from Selleck (Huston, TX, USA). 3-Methyladenine (3-MA), an inhibitor of autophagosomes, and Bafilomycin A1 (Baf-A1), an inhibitor of H⁺-ATPase, were purchased from Selleck. Antibodies against PARP (#9542), Drp1 (#5391), AMPKα (#5831), p-AMPKα (Thr172) (#2535), LC3 (#12741), Bak (#6947) and β-actin (#3700) were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies against Caspase 3 (#19677-1-AP), Caspase 9 (#10380-1-AP), Bcl2 (#12789-1-AP), Bcl-xl (#10783-1-AP), Bax (#50599-1-AP), Cytochrome c (#10993-1-AP), Aif (#17984-1-AP), Cox IV (#11242-1-AP), Fis1 (#10956-1-AP), Opa1 (#27733-1-AP), Mfn1 (#13798-1-AP), Ndufs1 (#12444-1-AP), Sdha (#14865-1-AP), Uqcrc1 (#21705-1-AP), Atp5a1 (#14676-1-AP), p62 (#18420-1-AP), and Beclin1 (#11306-1-AP) were obtained from Proteintech (Wuhan, China). Antibody against p-Drp1 (Ser616) (#12749) was obtained from Signalway Antibody (College Park, MD, USA). Secondary goat anti-rabbit or rabbit anti-mouse antibodies were purchased from Proteintech. Fluorescent-labeled antibody Annexin V-FITC, Annexin V-APC, PI, 7-AAD and 10 × binding buffer were obtained from BD (Franklin Lakes, New Jersey, USA).

Total protein in OC cells was extracted by a potent RIPA lysis reagent (Millipore, USA) containing 1% PMSF (Millipore, USA). The protein concentration of each sample was determined using the BCA method, and then samples (30 μg) were subjected to electrophoresis on an SDS-PAGE gel (Mellon, Dalian, China) and transferred onto PVDF membranes (Merck, USA). To prevent non-specific signals, the membranes were incubated with 5% BSA (Merck, USA) for 2 h. The primary antibodies, including LC3 (1:500; Cat No. 14600-1-AP, Proteintech, Wuhan, China), P62 (1:1000; Cat No. 18420-1-AP, Proteintech, Wuhan, China), FBXL11 (1:500; Cat No. 24311-1-AP, Proteintech, Wuhan, China), Beclin-1 (1:500; Cat No. 11306-1-AP, Proteintech, Wuhan, China), HA (1:5000; Cat No. 51064-2-AP, Proteintech, Wuhan, China), Flag (1:1000; Cat No. 80010-1-RR, Proteintech, Wuhan, China), and GAPDH (1:2000; Cat No. 60004-1-Ig, Proteintech, Wuhan, China) were added to incubate PVDF membranes at 4 °C overnight. After washing TBST for thrice, secondary antibodies were incubated for 2 h, and the protein signals in membranes were detected using the ECL reagent (Yeasen, Shanghai, China). GAPDH was set as a loading control to calculate the expression level of the target proteins.

For triple immunofluorescence staining (IF), the coverslips with cells were rinsed by using phosphate-buffered saline (PBS, pH 7.4), fixed in 4% paraformaldehyde for 20 min, incubated in 0.1% Triton-X100 for 5 min, and closed with 3% BSA for 2 h. Primary antibodies LC3 ӀӀ/Ӏ (ABclonal, China, 1: 200) and Beclin-1 (Proteintech, Chicago, IL, USA, 1: 500) were mixed, and the coverslips were hatched overnight at 4°C overnight, coverslips were incubated with mouse anti-CoraLite488-conjugated goat anti-mouse IgG (H+L) and rabbit anti-Cy3-conjugated Affinipure goat anti-rabbit IgG (H+L) for 2 h. DAPI (Boster, California, USA) was incubated for 10 min to localize cell nuclei, mounted with an anti-fluorescence quenching sealing liquid (Solarbio, Beijing, China), observed and imaged in Laser Confocal Microscope (LSM800, ZEISS, Oberkochen, Germany).

Western blot was performed as previously described [15 (link)]. The antibodies for the western blot were ATG3 (1:1000, Cell Signaling Technology, USA), ATG5 (1:1000, Cell Signaling Technology, USA), ATG7 (1:1000, Cell Signaling Technology, USA), ATG12 (1:1000, Cell Signaling Technology, USA), Beclin1 (1:1000, Proteintech, China), LC3 (1:1000, Novus, USA), p62 (1:1000, Proteintech, China), PGK1 (1:1000, Proteintech, China), Ubiquitin (1:1000, Proteintech, China) and β-actin (1:5000, Proteintech, China).

The whole knee joints of rats were collected and fixed in 4% paraformaldehyde for 24 h. After decalcification in 10% EDTA solution for 30 days, the samples were embedded in paraffin, cut into 5 μm–thick sections, and stained with H&E, Toluidine blue and Safranin O/Fast Green. The sections were next subjected to immunohistological staining using antibodies recognizing MMP13 (Abcam, 1;200), Cleaved-caspase3 (Cell Signaling Technology, 1:200) and Beclin1(Proteintech Group, 1:200). The Osteoarthritis Research Society International (OARSI) grade system were used to evaluate histopathologic changes in osteoarthritic cartilage [44 (link)]. All scorings were performed by three independent observers in a blinded manner. The rate of positive cells each section was quantitated from 6 rats of each group.