Adipogenic differentiation medium kit

The BMSCs at passage 3 in good growth state were detached and the concentration was adjusted into 5 × 10⁴ cells/mL, which were then inoculated into the 6-well plate pre-covered with cover glass. After 24 h, the BMSCs completely adhered to the wall. After that, BMSCs were incubated for 4 weeks using MSC Osteogenic Differentiation Medium Kit (Cyagen, Silicon Valley, CA, USA) and Adipogenic Differentiation Medium Kit (Cyagen, Silicon Valley, CA, USA). Next, the BMSCs were stained in order to determine the abilities of osteogenic and adipogenic differentiation in strict conformity to the protocols of the kits. Subsequently, the images were obtained under a microscope (CK40, Olympus, Tokyo, Japan). The markers of BMSCs were identified using flow cytometry. Antibodies fluorescein isothiocyanate (FITC)-CD105, FITC-CD73, phycoerythrin (PE)-CD90 (Abcam, Cambridge, UK), CD34, CD45 (PE, eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), and PE-HLA-DR (Abcam, Cambridge, UK) were added to different test tubes according to the specifications of antibodies, and immunoglobulin G (IgG) FITC or IgG (Abcam) served as the homologous control.

MSCs were plated in 6-well plate (1 × 10⁵ cells/well), and supplemented with appropriate differentiation medium for the induction of MSCs differentiation into different phenotypes. MSCs were cultured using OriCell^TM Balb/c Mouse Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation Medium Kit (Cyagen, Silicon Valley, CA, USA) and Adipogenic Differentiation Medium Kit (Cyagen, Silicon Valley, CA, USA) for 4 weeks, and staining was performed to confirm osteogenic and adipogenic differentiation ability according to the manufacturer's instructions provided by the kit. Then the images were captured using a microscope (CK40, Olympus, Tokyo, Japan).

3T3-L1 preadipocytes were purchased from a cell bank at the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, high-glucose, HyClone, USA) containing 10% fetal bovine serum (FBS, Invitrogen, USA). 3T3-L1 preadipocytes were transfected with the pLKD-CMV-G&PR-U6-shRNA vector and the PGMLV-CMV-KINDLIN2-HA-PGK-blasticidin vector which were purchased from OBiO Technology (Shanghai, China) and Genomeditech (Shanghai, China), respectively. The pLKD-CMV-G&PR-U6 vector and the PGMLV-CMV-HA-PGK-blasticidin were used as negative controls. The transfection efficiency was examined by western blot and real-time polymerase chain reaction (RT-PCR). For induction of adipogenic differentiation, cells were cultured in adipogenic differentiation medium kit (Cyagen, Suzhou, China). After being cultured for 21 days according to the protocol, cells were fixed in 4% paraformaldehyde solution for 30 minutes and then stained with Oil Red O (Cyagen, Suzhou, China) for 30 minutes at room temperature. For induction of osteogenic differentiation, cells were cultured in osteogenic differentiation medium kit (Cyagen, Suzhou, China). After being cultured for 21 days according to the protocol, cells were fixed in 4% paraformaldehyde solution for 30 minutes and then stained with Alizarin Red (Cyagen, Suzhou, China) for 5 minutes at room temperature.