Ab83259

After transfection, the BMSCs were fixed with 4% polyoxymethylene for 20 min at room temperature, permeabilized with 0.25% Triton X-100 (Sigma-Aldrich; Merck KGaA), and blocked with 0.1% bovine serum albumin (Roche Diagnostics) at room temperature for 30 min. Subsequently, the BMSCs were incubated overnight at 4°C with the following primary antibodies: Runx2 (Abcam, ab114133, 1:500), Osterix (Abcam, ab209484, 1:500), ALP (Abcam, ab83259, 1:500). After washing three times with PBS, the BMSCs were reacted with the appropriate secondary antibody (Bioss, Beijing, PV-0024, PV-0023, 1:100) for 30 min at 37°C. The cell nuclei were stained with haematoxylin (Abcam) at room temperature for 1 min and were visualized by light microscopy (magnification, ×200, Leica Microsystems GmbH). The BMSCs were observed at three different views to compare the degree of osteogenic differentiation between the different groups.

hDPSCs were lysed using RIPA buffer (Beyotime), and the concentration of extracts was quantified using the bicinchoninic acid (BCA) method. Samples (40 μg of protein) were fractionated by SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes (Millipore), followed by sealing with 5% nonfat milk for 1 hour. Thereafter, the membranes were incubated with primary antibodies at 4 °C for 24 hours and then reacted with secondary antibodies conjugated by HRP for 2 hours at 37 °C. Finally, an ECL reagent (Beyotime) was applied to quantify protein bands. The primary antibodies included B-cell lymphoma-2 (Bcl-2; ab59348), Cleaved Caspase-3 (c-caspase3; ab184787), Bcl2-Associated X (Bax; ab32503), runt-related transcription factor 2 (RUNX2; ab236639), alkaline phosphatase (ALP; ab83259), β-actin (ab8227; 1:1000) obtained from Abcam, and GIT2 (PA5-78301; Thermo Fisher Scientific).

Total protein from the BMSCs transfected with plasmids and siHOTAIR was isolated by RIPA lysis buffer (P0013B, Beyotime), and a BCA assay kit (23,250, Pierce, MA, USA) was used to detect the total protein concentrations. The total protein (30 µg) was separated in each lane on 10% SDS-PAGE gels (P0052A, Beyotime), electro-blotted and transferred to NC membranes (HTS112M, Millipore). Then, all the membranes were incubated by 5% skimmed milk for 2 hrs at normal atmospheric temperature, and followed by incubation with relative primary antibodies as follows: ALP (1:1000, 39kD, ab83259, Abcam), OCN (1:1000, 12kD, ab93876, Abcam), RUNX2 (1:1000, 37kD, ab76956, Abcam), LPL (1:1000, 25kD, ab91606, Abcam), PPARr (1:1000, 57kD, ab45036, Abcam), and GAPDH (1:1000, 37kD, ab181602, Abcam). The next day, HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000, ab205718, Abcam) or goat anti-mouse IgG secondary antibody (1:5000, ab205719, Abcam) was incubated with the membranes for 1 hr at room temperature. Finally, the membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate (34,078, Thermo Scientific, MA, USA) for signal detection. Image Lab™ Software (version 3.0) was used for densitometric analysis and quantification of the Western blot data (Bio-Rad Laboratories Inc., Hercules, CA, USA).