The MC3T3E1 cells and hOBs were cultured in DMEM containing 10 percent FBS, 100 mg/ml ascorbic p, nonessential amino acids and (-)-MK 801 penicillin/streptomycin. Cultures were maintained in a incubator at 37 C with five full minutes CO2. Immunofluorescence Cells grown on Lab Tek II Chamber Slides were fixed and incubatedwith an COX 2 goat polyclonal antibody and an anti p Akt rabbit polyclonal antibody. Phycoerythrin conjugated anti goat and fluorescein conjugated anti rabbit secondary antibodies allowed creation of COX 2 and r Akt, respectively. All cells were stained with DAPI for nuclear declaration. Cells were captured and then visualized by confocal fluorescence microscopy. Ahead of siRNAtransfection,we used the BLOCK iT Alexa Fluorred fluorescent get a handle on as an indicator 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 Papillary thyroid cancer transfection Cells a general RNAi negative control were cultured in Opti MEM during siRNA transfection, after which the medium was replaced with complete culture medium. After 24 h, mRNAexpression, protein amounts or phosphatase activitywere reviewed. Cells were transfected with 100U rhCOX 2 protein using the Pro Ject protein transfection reagent in Opti MEM. For the lazy rhCOX 2 protein transfection party, 100U rhCOX 2 was incubated with 10 uM NS398 for 1 h at 37 C just before protein transfection. After transfection, culture medium was replaced with complete culture medium, and after 24 h, the cells were collected for protein analysis. After thehOBswere Gossypol molecular weight transfectedwith siRNA, totalmRNAwas separated using TRIZOL reagent. Quantitative realtime PCR was done with a Bio Rad iQ5 real-time PCR detection system utilising the iQ SYBR green supermix. The precise PCR services and 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 given as a value of one, and the gene expression degree of each experimental group was calculated relative to the control. After siRNA and/or rhCOX 2 protein transfection, cells were incubated with recombinant human IGF for 20 min and then lysed in the PhosphoSafe Reagent for protein removal. Mobile lysates containing 50 ug of proteins were analyzed by ten percent SDS PAGE. Transferred membranes were incubated with antibodies against COX 1, Akt, GSK 3/B, FOXO1, PTEN, complete phosphorylated PTEN, COX 2, p27Kip, g Akt, phosphorylated Gsk 3/B, FOXO3a, Ser380 phosphorylated PTEN, or B actin.