P pi3k

AQX-1125 was purchased from MedChemExpress (MCE). The primary antibodies of GAPDH, PI3K, p-PI3K, Akt, p-AKT, P65, p-P65, IκBα, p- IκBα, SHIP1, Runx2, Alp, NFATc1, and c-Fos were acquired from ABclonal (Wuhan, China). Phalloidin and 4, 6-diamidino-2-phenylindole (DAPI) were purchased from Solarbio (Beijing, China). RANKL (the receptor activator of the nuclear factor kappa-B ligand) and M-CSF (macrophage colony-stimulating factor) were obtained from R&D Systems (Minnesota, USA). Cell culturing plates were purchased from NEST (Jiangsu, China). Minimum Essential Medium Alpha (α-MEM), Dulbecco’s Modified Eagle Medium: F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin were purchased from Gibco (Grand Island, NY, United States). The TRAP staining kit was obtained from Solarbio (Beijing, China).

UP302 was purchased from Nanjing m&m biotechnology Co. Ltd. The following reagents were used: fetal bovine serum (Procell, China), Dulbecco's Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco's Medium(IMDM), trypsin (Servicebio, China), CCK8 (Sparkjade, China), chloroquine (Sparkjade, China), PI and FITC, Mitochondrial membrane potential assay kit with JC-1 and Reactive Oxygen Species Assay Kit (Beyotime, China), Antibodies against Caspase-3, BAX, BCL-2, LC3B, AKT123, PI3K, P-PI3K, P-AKT, AIF, Tomm20, PINK1, Parkin, Beclin1, and β-actin were purchased from ABclonal(China). Twelve Balb/c female nude mice, aged four weeks, were purchased from Jinan Pengyue Experimental Animal Breeding Co. LTD.

Cells or tissues were lysed in RIPA buffer and protein concentration was determined by BCA protein assay. Nuclear extraction from total lysis was performed using Subcellular Structure Cytoplasmic and Nuclear Extraction Kit. Then, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were electrotransferred to PVDF membrane (Millipore, Billerica, USA) and then blocked with 5% non-fat milk for 4 h. The primary antibodies against DSC2 (#53485, Santa Cruz Biotechnology, USA), p-PI3K (#0854, ABclonal Technology, Wuhan, China), PI3K (#4255, Cell Signaling Technology, Massachusetts, USA), p-AKT (#66444, Proteintech, Wuhan, China), Akt (#60203, Proteintech, Wuhan, China), Cyto-C (#4280, Cell Signaling Technology, Massachusetts, USA), caspase-3 (#66470, Proteintech, Wuhan, China), BCL-2 (#12789, Proteintech, Wuhan, China), p53 (#AF0879, Affinity Biotechnology, USA), PTEN (#60300, Proteintech, Wuhan, China), γ-catenin (#66445, Proteintech, Wuhan, China), Lamin B1 (internal reference for the nucleus, #AF5161, Affinity Biotechnology, USA), and β-actin (internal reference for the total cell, #ZF-0313, ZS Bio, Beijing, China) were used.

Total protein was extracted from cells with RIPA lysis buffer (Beyotime, P0013B, China) containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and various inhibitors (sodium orthovanadate, sodium fluoride, EDTA, leupeptin, etc.), and then quantified with a BCA Protein Assay Kit (Thermo Scientific, USA). A 30 µg denatured protein sample was separated by 10–12% SDS–PAGE and was then transferred to PVDF membranes (Millipore). After blocking in 5% nonfat milk for 2 h at room temperature, the membranes were incubated with primary antibodies specific for COL1A1 (1:1000, Abcam, UK), PI3K (1:500, Abclonal Technology, USA), p-PI3K (1:500, Abclonal Technology, USA), AKT (1:500, Abclonal Technology, USA), and p-AKT (1:500, Abclonal Technology, USA) overnight at 4 °C. Thereafter, incubation with the secondary antibody was conducted for an additional 1 h at room temperature. Finally, an ECL reagent was employed for protein visualization (Millipore, USA). GAPDH was employed as the internal control.