At sub confluence, cells were detached with trypsin and at the third passage the cells were plated in a 96 well cell culture plate or 25 ml cell culture flasks at a density of 3. 0 �� 105 cells ml. The cells were cultured in osteogenic differentiation medium consisting of DMEM with 10% FBS supplemen ted with 10 mM b glycero phosphate and 0. 1 mM L ascorbic 2 phosphate. MLO Y4 osteocytes, a either cell line derived from mouse long bones, was cultured in minimum essential medium supplemented with 2. 5% fetal bovine serum, 2. 5% calf serum, 100 U ml streptomycin and 100 ug Inhibitors,Modulators,Libraries ml penicillin at 37 C and 5% CO2 in air. The cells were cultured in osteogenic differentiation medium consisting of MEM with 2. 5% fetal bovine serum, 2. 5% calf serum, 100 U ml streptomycin and 100 ug ml penicillin supplemented with 10 mM b glycero phosphate and 0.

1 mM L ascorbic 2 phosphate. The cultures were treated with Ris at concentrations ranging from 0. Inhibitors,Modulators,Libraries 1 to 10 uM with or without NS 398, or with dexametasone as specified in the figures, and controls were treated with vehicle. XTT assay Cell viability was evaluated by a colorimetric assay based on the reduction Inhibitors,Modulators,Libraries of the tetrazolium salt XTT benzene sulfonic acid hydrate by mito chondrial dehydrogenase of viable cells to a formazan dye. Briefly, cells were cultivated in 96 microtitre plates in medium con taining different concentrations of Ris with or without NS 398. After 72 h, 50 ul XTT labeling mixture was added to each well and incubated at 37 C for 4 h. The spectrophotometric absorbance of the samples was mea sured using a microtitre plate reader at a wave length of 450 nm.

Total RNA extraction and reverse transcription Inhibitors,Modulators,Libraries Total RNA was extracted from each cell pellet using the RNAeasy minikit with DNAse I treatment. The amount and purity of the RNA were checked by measuring the absorbance at 260 and 280 nm, and where a ratio ranging from 1. 8 to 2. 0 was taken to be pure. First strand cDNA was generated from 1 ug of total RNA using the First Strand cDNA Synthesis Kit, with random hexamers, according to the manufacturers pro tocol. RT product was aliquoted in equal volumes and stored at 80 C. Real time RT PCR mRNA quantification was analyzed by Relative Standard Curve Method. PCR was performed in a total volume of 25 ul containing 1 �� Taqman Universal PCR Master mix, no AmpErase UNG and 5 ul of cDNA from each sample, pre designed, Gene specific primers and probe sets for each gene were obtained from Assay on Demande Gene Expression Products Applied Inhibitors,Modulators,Libraries Biosystems.

The Real Time amplifications included 10 minutes at 95 C, followed truly by 40 cycles at 95 C for 15 seconds and at 60 C for 1 minute. Thermocy cling and signal detection were performed with ABI Prism 7300 Sequence Detector. Signals were detected according to the manufacturers instructions and we selected the Rn in the exponential phase of amplification plots to determine the Ct values and to obtain the linearity of calibration curves.