Bcl2 associated x (bax)

After 48 h of HEA treatment, SGC-7901 cells were collected, total protein was extracted, and Western blot analysis was performed as previously described [13 (link)]. Primary rabbit antibodies against β-actin (1:3000), AIF (1:2000), Bcl-2 (1:2000), and Bax (1:2000) were purchased from HUABIO (Hangzhou, China). Primary antibodies against caspase-3 (1:2000), caspase-8 (1:2000), caspase-9 (1:2000), caspase-12 (1:1000), cytochrome C (1:500), poly(ADP-ribose) polymerase (PARP) (1:2000), cleaved-PARP (1:2000), and p53 (1:1000), were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against ATF4 (1:1000), CHOP (1:500), p62 (1:1000), and Beclin1 (1:1000) were obtained from Proteintech (Rosemont, IL, USA). Primary antibodies against LC3 (1:500), ATG5 (1:1000), and ATG12 (1:1000) were obtained from MBL (Nagoya, Japan). Anti-rabbit lgG horse-radish peroxidase-conjugated secondary antibody (1:3000) was purchased from Abcam (Cambridge, MA, USA).

Murine ovarian tissue and drug-treated KGN cells were subjected to protein extraction in RIPA lysis and extraction buffer (Thermo, USA). Protein concentrations were quantified using the BCA protein assay kit (Thermo, USA). The protein samples were fractionated by SDS‒PAGE and then transferred to PVDF membranes (Millipore, USA). Subsequently, the membrane was blocked using 5% skim milk for 1 h. Then, the sections were incubated overnight at 4 °C with the corresponding primary antibodies, including pATM, PGC-1α, TERF2 (ABclonal, China), SIRT1, p53 (Proteintech Group, China), Bax, Bcl-2, and β-actin (HuaBio, China), and then probed with secondary antibodies. Protein chemiluminescence was detected using the ECL chemiluminescence reagent (Thermo, USA). Finally, exposure was performed using an exposure device.

Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China, P0013B). Total protein was quantified by BCA protein assay (Bio‐Rad, Berkery). Each sample was adjusted to equal amount of protein using 5X loading buffer for loading. The samples were separated by SDS‐PAGE, transferred to a polyvinylidene fluoride membrane, and immunoblotted with the following antibodies: GDF11 (DGDF80, R&D Systems,Emeryville, CA,USA), HGF (ab83760, Abcam, USA), VEGFR1 (ET1605‐11,Huabio，China), CD31 (#77699, Cell Signalling TechnologyBoston,MA, USA), VEGFR2 (#9698, following Abs are all from Cell Signalling Technology,USA), phospho‐p44/42 (Thr202/Try204 phospho‐ERK1/2,#4370), p44/42 MAPK (Erk1/2,#4696), BCL2 (#2827), BAX (#14796), Cleaved Caspase3 (#9661), phospho‐Smad2 (Ser465/Ser467,#18338), phospho‐Smad3 (Ser423/425, # 9520), Smad2(#5339), Smad3(#9523), anti‐β‐actin (#3700), EIF4E (R1512‐8,Huabio, China), Phospho‐eIF4E (S209) (ET1608‐66,Huabio, China) and VEGF ( ER30607,Huabio, China),at 4°C overnight. After incubation of the membranes with peroxidase‐conjugated secondary antibodies (Cell Signalling Technology,USA), bands were visualized using enhanced chemiluminescence reagents (Bio‐Rad,Los Angeles, CA,USA).

A 500 mM DSF (Sigma-Aldrich; Merck KGaA) stock solution was dissolved in high-quality anhydrous dimethyl sulfoxide (DMSO) and stored in the dark at − 20 °C. A stock solution of copper gluconate (Sigma-Aldrich; Merck KGaA) at a concentration of 500 μM was prepared by dissolving the compound in ddH₂O. A 500 mM N-acetyl-l-cysteine (NAC; Sigma-Aldrich; Merck KGaA) stock solution was dissolved in high-quality anhydrous DMSO and stored in the dark at − 20 °C. Antibodies for western blot analysis were as follows: caspase 3 (cell signaling technology), β-actin (ProteinTech), AIP (cell signaling technology), p53 (cell signaling technology), BCL6 (cell signaling technology); cleaved (c)-caspase 3 (Abcam), BCL2 (Abcam), BCL-XL (Abcam), IκB (Abcam), phosphorylated (p)-IκB (Abcam), NF-κB p65 (Abcam) and BAX (HuaBio). Antibodies for flow cytometry were as follows: Alexa Fluor^® 647-conjugated BCL2 (clone Bcl-2/100; BD Pharmingen™; BD Biosciences), Alexa Fluor^® 488-conjugated BAX (clone 2D2; BioLegend.), PE-Cy™7-conjugated BCL6 (clone K112-91; BD Biosciences) and PE-conjugated BCL-XL (clone 7B2.5; Invitrogen; Thermo Fisher Scientific, Inc.). The Cell Counting Kit-8 (CCK-8) was purchased from Genview. JC-1 Mitochondrial Membrane Potential Detection Kit (C2006) was purchased from Beyotime Institute of Biotechnology. Dihydrorhodamine (DHR) 123 (D1054) was purchased from Sigma-Aldrich; Merck KGaA.

The total proteins from different macrophages and chondrocytes were extracted with the RIPA lysis buffer (MCE, NJ, USA) containing a mixture of phosphatase and protease inhibitors. Then, the proteins were separated with 8–12% SDS-PAGE (Febio science, Hangzhou, China) and transferred to the polyvinylidene fluoride membranes (Merk Millipore, Darmstadt, Germany). The membranes were blocked with 5%BSA and then incubated with the primary antibodies overnight. The membranes were washed next day with PBST and then incubated with the HRP-conjugated secondary antibodies (Beyotime, Shanghai, China) for 90 min. These results on the membranes were captured and documented by Molecular Imager (Bio-Rad, CA, USA).
The primary antibodies used include those against IL-1β (R&D Systems, MN, USA), NLRP3 (Adipogen, CA, USA), ASC (Adipogen), Caspase-1 (Adipogen), P-jak2 (Abcam, MA, USA), P-Stat3 (Abcam), P-bad (Cell Signaling Technology, MA, USA), Cleaved Caspase-3 (Cell Signaling Technology), Jak2 (ABclonal, Wuhan, China), Stat3 (ABclonal), Bad (ABclonal), Clic1 (ABclonal), Clic4 (ABclonal), ADAMTS5 (ABclonal), MMP13 (ABclonal), Bax (HUABIO, Hangzhou, China), Bcl-2 (HUABIO), Clic5 (HUABIO), PIM-1 (HUABIO), Collagen II (ProteinTech, Wuhan, China), Aggrecan (ProteinTech), β-actin (ProteinTech), GADPH (ProteinTech).