Apelin

Total protein was extracted from cells and tissues, as well as protein concentration was determined using a BCA detection kit (Boster Biotech, Wuhan, China). Proteins were separated by electrophoresis on a sulfuric acid dodd sodium-polyacrylamide gel and transferred to a polyvinylidene fluoride membrane. Five percent thin milk powder blocked the membranes for 2 h at room temperature, with antibodies incubating to Apelin (1:1000, Abcam, Cambridgeshire, England), Beclin 1 (1:1000, Cell Signaling Technology, Danvers, MA, USA), Bax (1:1000, Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (1:1000 Abcam, Cambridgeshire, England), cleaved-caspase3 (1:500 Abcam, Cambridgeshire, England), caspase3 (1:5000 Abcam, Cambridgeshire, England) and β-actin (as a gel-loading control, 1:1000, Cell Signaling Technology, Danvers, MA, USA) was incubated at 4 °C for 12 h. Membranes were incubated with HRP-conjugated secondary antibody (1:5000, Dingguo, Beijing, China) at room temperature for 2 h, and then immunolabeled bands were visualized using enhanced chemiluminescence reagent (Beyotime Biotech, Hangzhou, China). Protein expression levels were detected by ImageJ software (NIH, Bethesda, MD, USA).

Cells were lysed in RIPA buffer combined with proteasome inhibitor and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then separated target proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 60 min, the membranes were incubated overnight at 4 °C with antibodies specific to GAPDH (1:2000; Abcam, Shanghai, China), APELIN (1:2000; Abcam, Shanghai, China), RUNX2 (1:1600; Abcam, Shanghai, China), COL1A1 (1:1000; Abcam, Shanghai, China), non-phosphorylated (active) β-catenin (1:1000; Abcam, Shanghai, China), or total β-catenin (1:1000; Abcam, Shanghai, China). After washing with TBST three times (10 min each), the membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated, Beyotime) for 1 h at room temperature. After washing three times with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured by Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

Cells were cultured in a 12-well plate with induction medium and evaluated for RUNX2 and APELIN using a fluorescence microscope (EU5888; Leica, Germany) as follows. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized for 30 min in 0.2% Triton X-100, and blocked for 30 min in 2% bovine serum albumin. Fixed cells were washed and incubated overnight with anti-RUNX2 (1:1600; Abcam, Shanghai, China), APELIN (1:2000; Abcam, Shanghai, China), or COL1A1 (1:1000; Abcam, Shanghai, China). After washing three times with ddH2O, cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) for 1 h, the nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 5 min. Target proteins were observed under a fluorescence microscope (Leica).