C3h10t1 2

C3H10T1/2, MC3T3-E1, and 3T3-L1 cells were purchased from American Type Culture Collection. C3H10T1/2 and 3T3-L1 cells were maintained in DMEM (GIBCO-BRL, CA, USA), whereas MC3T3-E1 cells were maintained in MEMα (catalog no. A1049001, GIBCO-BRL, CA, USA). Culture media were supplemented with 10% fetal bovine serum, glutamine, penicillin, and streptomycin. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. C3H10T1/2 cells were transfected with pcDNA3.1-ZFP36L1 and selected with G418 (1 mg/ml). Twenty two antibiotic-resistant clones were harvested and examined for ZFP36L1 overexpression. Clones 2 and 21 which expressed high level of ZFP36L1 were selected for experiments. The femurs and bone marrow mesenchymal stem cells isolated from the femurs of adult and aged Fisher 344 rats were kind gifts from Dr. Chun-Chin Liang. For the isolation of RNA from rat femurs, the femurs were cryogenically pulverized at -195 °C by liquid N2 using a SPEX 6770 Freezer/Mill (SPEX SamplePrep, NJ, USA). Total RNA was then extracted using Trizol Reagent (Life Technology, MD, USA).

MC3T3-E1, C3H10T1/2, and C2C12 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). C2C12 and C3H10T1/2 cells were maintained in DMEM (Gibco, Grand Island, NY, USA) while MC3T3-E1 cells were cultured in α-minimal essential medium (α-MEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). For differentiation experiments, MC3T3-E1 cells were seeded at density of 1×10⁴ cells/cm² with growth media (GM, α-MEM containing 10% FBS). After 24 h of incubation, cells were exposed to indicated concentrations of Edil3 in GM or osteogenic media (OM) containing 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate. As control for osteogenic differentiation, 200 ng/ml recombinant human bone morphogenetic protein 2 (BMP-2, Osstem implant, Seoul, Korea) in growth media was used for cell culture.