Evaluation of mineralization Mineralization of cell cultures was evaluated by alizarin red S staining. OBs were seeded at 2 inhibitor Bosutinib 105 cells well in six well tissue culture dishes and maintained in MEM, 10% FBS supplemented with 10 mM B glycerophosphate, at 37 C in a humidified atmosphere containing 5% CO2. Culture medium was replaced every 3 days until day 20. OBs were treated with MSU or ve hicle at day 8. At day 20, cells were fixed for 20 minutes with buffered formalin and then stained for 20 minutes with 40 mM ARS, pH 4. 0 to 4. 2 at room temperature. After four washes with distilled H2O, ARS was extracted, as previously described. In brief, ARS cells were incubated 30 minutes with acetic acid and then heated 10 minutes at 85 C, pH was re stored at 4. 2 with NaOH, and ARS absorbance was read at 405 nm.

MMP activity Evaluation of generic matrix metalloproteinases Inhibitors,Modulators,Libraries was assessed with the SensoLyte Generic MMP assay kit that detects the activity of a variety of MMPs, including MMP 1, 2, 3, 7, 8, 9, 12, 13, and 14. Five FAM and QXL520, labeled FRET peptide substrates, were used for continuous measurement of the enzyme activities. On the cleavage of the FRET peptide by MMPs, the fluorescence of 5 FAM was recovered and monitored at excitation emission wavelengths of 490 nm Inhibitors,Modulators,Libraries 520 nm. Confluent OBs were treated 24 hours with or without 0. 5 mg MSU in MEM containing 1% FBS. Medium was then centrifuged 2 minutes at 10,000 rpm, and 50 ul of supernatant was added to 50 ul of MMP substrate for 20 minutes. MMP activity in MSU stimulated cells was compared with MMP activity in untreated cells.

RNA isolation and real time PCR OB total RNA was isolated by using Trizol. In brief, around Inhibitors,Modulators,Libraries 106 confluent cells, stimulated with MSU or vehicle, were washed in PBS and then homogenized in 1 ml Trizol. Total RNA was then extracted, according to the manufacturers Inhibitors,Modulators,Libraries protocol. Reverse transcription and real time PCR were performed essentially as previously described in. In brief, first strand cDNA synthesis was performed Inhibitors,Modulators,Libraries by using 1 ug of total RNA with Superscript II in recommended conditions, with 10 ng of random hex amers. Amplification of osteoblast cDNA was carried out in a Rotor Gene 3000 operated with Rotor Gene software version 6. 0. 19. Each sample consisted of, 50 ng cDNA, 1. 3 mM MgCl2, 0. 2 mM dNTP, 500 nM primers, 0. 5 unit of Taq polymerase, and Sybr Green dye in a reaction www.selleckchem.com/products/nutlin-3a.html volume of 20 ul. Amplification conditions were as follows, 95 C, 60 C, 72 C, 35 cycles. Specificity of each reaction was ascertained by performing the Melt procedure after completion of the amplification protocol, according to the manufacturers instructions.