Plkd cmv g pr u6 shrna vector

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.