Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Article Title: A NOVEL, DIRECT NO DONOR REGULATES OSTEOBLAST AND OSTEOCLAST FUNCTIONS AND INCREASES BONE MASS IN OVARIECTOMIZED MICE
Figure Lengend Snippet: NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation, and survival in POBs (A) POBs isolated from intact C57Bl6 mice were incubated in medium with 0.1% FBS (3 × 10 5 cells/ml) for 2 h prior to receiving vehicle or 10 μM NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NO x ) were measured in the medium by the Griess reaction (NO x present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min. The intracellular cGMP concentration was measured by ELISA (B), and VASP phosphorylation was analyzed by Western blot using a phospho-Ser 259 -specific antibody, with bar graph showing densitometric quantification of pVASP normalized to β-actin (C). (D,E) Serum-deprived POBs were treated with vehicle or 10 μM NO-Cbi for 10 min and ERK ( D ) and Akt ( E ) activation were assessed by blotting with phospho-specific antibodies. Densitometric quantification of pErk and pAkt normalized to total Erk and Akt, respectively, is shown in the bar graphs. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μM NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs were cultured in medium with 0.1% FBS for 18 h, and treated with 10 μM NO-Cbi or vehicle for 1 h; they were transferred to fresh medium containing 3 H-thymidine for 24 h, and thymidine incorporation into DNA was measured. ( H,I ) Confluent POBs were differentiated in ascorbate-containing medium for 14 d, with some cells receiving 10 μM NO-Cbi (filled bars) or vehicle (open bars) during the last 24 h. Expression of osteocalcin (OCN), osteopontin (Spp1), collagen 1-A1 (Col1a1), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tuba1), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNA was determined by qRT-PCR and normalized to 18S rRNA; normalized mRNA in vehicle-treated cells was assigned a value of 1. Panels A–I show means ± SEM of at least three independent experiments; *p
Article Snippet: To generate PRKG2f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation).
Techniques: Expressing, Isolation, Mouse Assay, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Immunofluorescence, Staining, Cell Culture, ALP Assay, Quantitative RT-PCR