C jun sirna

MC3T3-E1 cells were seeded into plates in complete medium and cultured for 24 days until the cells reached 70% confluence. To initiate the differentiation, the cells were incubated in osteogenic induction medium (OIM) containing α-MEM, 10% FBS, dexamethasone (10-7M), β-glycerophosphate (10 mM) and ascorbic acid (50 μg/ml). The differentiation medium was replaced every 3 days, with DMSO, BDNF (50 ng/ml), or 7,8 DHF (0.5 μM) added into the medium with or without the pretreatment of K252a (100 nM). The MC3T3-E1 cells were transfected with AEP C189S, AEP WT, AEP T322E plasmid, C/EBP β siRNA (sc-29862, Santa Cruz Biotechnology, USA), CREB siRNA (sc-35111, Santa Cruz Biotechnology, USA), C-Jun siRNA (sc-29224, Santa Cruz Biotechnology, USA) or control plasmid or control-siRNA (sc-44237, Santa Cruz Biotechnology, USA) by Lipo3000 transfection reagent according to the instructions.

siRNA against c-Myb was synthesized in vitro using a Silencer® siRNA Cocktail Kit (RNase III) (Invitrogen, California, USA) as per the manufacturer’s instruction. Briefly, using c-Myb-specific primers having a T7-promoter leader (8 nucleotides) sequence (Additional file 1: Table S3), a c-myb-specific stretch of cDNA was amplified from LSK-CD34⁻ cells. Amplicons were again amplified using T7 promoter primer. The sense and antisense siRNA templates were transcribed by T7 RNA polymerase and the resulting RNA transcripts were hybridized to create dsRNA. RNAse III was used to produce siRNAs (12–30 base pairs long) which were further purified. The resulting c-myb siRNA or c-jun siRNA (Santa Cruz Biotech, TX, USA) were transfected into sort-purified LSK-CD34⁻ cells using Dharmafect reagent (Thermo Scientific, MA, USA) in a 1:1 ratio. Mock transfected cells were used as controls. Efficiency of silencing of these SiRNA was determined by qRT-PCR using c-Myb- and c-Jun-specific primers. Cells were incubated for 12–18 h and then treated with SNP for 12 h. Levels of c-Jun, c-Myb, and Cd34 mRNA were analyzed by qRT-PCR.