Enhanced chemiluminescence reagent

Cells for Western blotting were treated with DMSO or PEITC for 24 h. Total cell proteins were extracted using RIPA lysis buffer containing protease and phosphatase inhibitors. A BCA protein assay kit (Solarbio, China) was used to detect the protein concentrations. 30 μg total proteins were loaded on an SDS-gel, separated using SDS-PAGE, and then transferred to PVDF membranes (Merck Millipore, MA, United States of America). Membranes were subsequently blocked in 5% skimmed milk at ambient temperature for 2 h, followed by incubation with anti-GAPDH (Proteintech, #10494-1-AP), anti-p53 (abcam, #ab131442), anti-cleaved caspase 3 (CST, #9664), anti-PAb240 (Merck Millipore, #OP29L), anti-PAb1620 (Merck Millipore, #OP33), anti-p21 (CST, #2947), anti-Bcl2 (Proteintech, #12789-1-AP), anti-BAX (CST, #14796), anti-PUMA (Proteintech, #55120-1-AP), anti-MDM2 (Proteintech, #27883-1-AP), anti-CyclinB1 (abcam, #ab32053) or anti-p73 (abcam, #ab215038) overnight at 4°C. The membranes were then rinsed in TBST for 20 min and incubated for 1 h with the secondary antibody (Affinity Biosciences, Cincinnati, OH, United States) at room temperature. Signals were detected using enhanced chemiluminescence reagent (Affinity Biosciences, Cincinnati, OH, United States) and imaged using an AI600 series imager (GE Healthcare, Carlsbad, CA, United States).

Osteoblast cells were seeded into six-well plates at a density of 1 × 10⁶ cells/well. After stimulation was complete, cells were treated with RIPA lysis buffer (Bioteke) with 1 mm phenylmethylsulfonyl fluoride (PMSF) (Solarbio), 1% phosphatase inhibitors (Roche, Basel, Switzerland), and 1% proteinase inhibitor (Roche) for 30 min on ice. Cell lysates were resolved using 8–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis followed by transfer to polyvinylidene fluoride (PVDF) membranes. Membranes were then blocked with skim milk (Solarbio) at room temperature for 1 h and then incubated with the appropriate primary antibodies against RANKL, GAPDH, p-GP130, p-STAT3, STAT3, p-JAK2, or JAK2 (Affinity, USA) on an orbital shaker at 4°C overnight. Then, membranes were washed (5 min, three times) with tris-buffered saline with 0.1% Tween and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies diluted 1: 6,000. After washing (5 min, three times), membranes were incubated with an enhanced chemiluminescence reagent (Affinity), and the chemiluminescent signals were visualized using an iBright CL1000 imaging system (Thermo, MA, USA). The semiquantitative analysis was performed using ImageJ v1.8.0 (NIH, MD, USA).

JC105 was synthesized in our laboratory (see below). Isosteviol was purchased from Sigma–Aldrich Co. Ltd. U0126 was purchased from Cell Signaling Technology. 3‐[4, 5‐dimethylthiazol‐2‐yl] 2, 5‐diphenyl‐tetrazolium bromide (MTT) was purchased from VWR Life Sciences. Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), 0.05% Trypsin‐EDTA (1×), penicillin (10 000 U/mL)‐streptomycin (10 000 µg/mL) (Pen Strep), phosphate‐buffered saline (PBS, 1×), no glucose and no sodium pyruvate DMEM were purchased from Gibco. RIPA buffer was obtained from Biosharp. Protease inhibitor (100x) was obtained from Beyotime. Bicinochoninic acid (BCA) assay kit was obtained from Beyotime. Stock concentration of JC105 (100 mM) and U0126 (10 mM) was dissolved in DMSO. MTT (5mg/ml) was dissolved in DMEM. During all treatments, the concentration of DMSO was below 0.1% (v/v). Phospho‐ERK 1/2 (Thr²⁰²/Tyr²⁰⁴), Total‐ERK1/2, Phospho‐DRP1 (Ser⁶¹⁶), Total‐DRP1, Phospho‐AKT (Ser⁴⁷³) and Total‐AKT were obtained from Cell Signaling Technology. Phospho‐AMPKα (Thr¹⁷²) and Total‐AMPK were obtained from Abcam and GAPDH was obtained from Proteintech. Anti‐rabbit IgG and anti‐mouse IgG were obtained from Proteintech. Enhanced chemiluminescence reagent was obtained from Affinity Biosciences.