Mir 23b inhibitor

The miRNA mimics (miR-23b mimic), inhibitors (miR-23b inhibitor) and negative controls of miR-23b were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China) and transfected into C2C12 at final concentrations of 50 nM per well in a 24-well plate with Entranster-R transfection reagent (Engreen, Beijing, China) following the manufacturer's instructions. The component was mixed in serum-free DMEM, and then the transfection was conducted in complete DMEM and refreshed 6 h subsequent to transfection. The plasmids were transfected into cells using Lipofectamine 2000 Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) as described previously (14 (link)).

Scramble miRNA, miR-23b mimic, miR-23b inhibitor, negative control of miR-23b inhibitor, programmed cell death 4 (PDCD4)-specific small interfering RNA (si-PDCD4), and its negative control (siNC) were all purchased from GenePharma Co. (Shanghai, China). The full-length PDCD4 was ligated into a pEX-2 plasmid (GenePharma Co.), and the recombined plasmid was referred to as pEX-PDCD4. Lipofectamine 3000 regent (Invitrogen) was utilized for cell transfection in line with the protocol of the manufacturer. The empty pEX-2 plasmid was also transfected into ATDC5 cells, termed as pEX group. Stable transfection was selected by G418 (0.5 mg/ml; Sigma-Aldrich). Cells transiently transfected with miRNAs were harvested at 72 hr post-transfection.

Rat aortic smooth muscle cell (RA-SMC) line was purchased from ScienCell Research Laboratories and cultured (at 37°C, 5% CO₂) in Dulbecco’s modified Eagle medium (GIBCO, LifeTechnologies, USA), containing 10% FBS (GIBCO, LifeTechnologies, USA) with 1% of penicillin-streptomycin. Cells were treated with different concentrations of platelet-derived growth factor-BB (PDGF-BB) for different lengths of time. XR007793 siRNA, miR-23b mimic, and miR-23b inhibitor and were purchased from Genepharma Company (Shanghai, China). Cells at 70% confluence were transfected with siRNA or miRNA mimics/inhibitor by Lipofectamine 2000 reagent (Life Technologies, USA). Antibodies against FOXO4 antibody (ab128908, 1: 5000), SMα-actin antibody (ab5694, 1: 5000), SM22 antibody (ab10135, 1: 5000) were purchased from Abcam (Cambridge, MA, USA), secondary antibody (SA00001-2, 1: 2000) was purchased from Proteintech (Wuhan, China)