The MC3T3E1 cells and hOBs were cultured in DMEM containing 10 percent FBS, 100 mg/ml ascorbic acid, nonessential amino acids and Clindamycin clinical trial penicillin/streptomycin. Cultures were maintained in a incubator at 37 C with 5% CO2. Immunofluorescence Cells grown on Lab Tek II Chamber Slides were fixed and incubatedwith an COX 2 goat polyclonal antibody and an anti g Akt rabbit polyclonal antibody. Phycoerythrin conjugated anti goat and fluorescein conjugated anti rabbit secondary antibodies allowed visualization of COX 2 and p Akt, respectively. All cells were stained with DAPI for nuclear declaration. Cells were then visualized and photographed by confocal fluorescence microscopy. Just before siRNAtransfection,we used the BLOCK iT Alexa Fluorred fluorescent control as an sign of the transfection efficiency of hOBs using the Lipofectamine RNAiMAX reagent. Cells were transfected with COX 1 siRNA, COX 2 siRNA No. 1, PTEN siRNA, COX 2 siRNA No. 2 or as a for siRNA Chromoblastomycosis transfection Cells a common RNAi negative control were cultured in Opti MEM throughout siRNA transfection, after which the medium was replaced with complete culture medium. After 24 h, mRNAexpression, protein amounts or phosphatase activitywere examined. Cells were transfected with 100U rhCOX 2 protein using the Pro Ject protein transfection reagent in Opti MEM. For the inactive rhCOX 2 protein transfection party, 100U rhCOX 2 was incubated with 10 uM NS398 for 1 h at 37 C ahead of protein transfection. After transfection, culture medium was changed with complete culture medium, and after 24 h, the cells were obtained for protein analysis. After thehOBswere mapk inhibitor transfectedwith siRNA, totalmRNAwas separated using TRIZOL reagent. Quantitative realtime PCR was performed with a Bio Rad iQ5 real-time PCR detection system utilizing the iQ SYBR green supermix. The precise PCR products were detected by measuring the fluorescence of SYBR Green, a strandedDNA binding dye. The relativemRNAexpression levelwas normalized toGAPDH. Themean of the relative value of gene expression in the control group was assigned as a value of one, and the gene expression level of each experimental group was calculated relative to the control. After siRNA and/or rhCOX 2 protein transfection, cells were then lysed in the PhosphoSafe Reagent for protein removal and incubated with recombinant human IGF for 20 min. Mobile lysates containing 50 ug of proteins were analyzed by ten percent SDS PAGE. Shifted membranes were incubated with antibodies against COX 1, Akt, GSK 3/B, FOXO1, PTEN, full phosphorylated PTEN, COX 2, p27Kip, r Akt, phosphorylated Gsk 3/B, FOXO3a, Ser380 phosphorylated PTEN, or B actin.