Culture of primary osteoblast cells and osteoblast cell lines Osteoblastic cell lines: C2C12 (ATCC, #CRL-1772), 2T3 [54], UMR106 (ATCC, #1661), and MC3T3-E1 (ATCC, #2593) cells were seeded into 6 to 48-well plates with complete culture media (DMEM for C2C12 and UMR106 cells and aMEM for 2T3 and MC3T3-E1 cells), supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin and 1% L-Glutamine, at a cell density that allowed cells to reach 60–70% confluence after overnight culture in 5% CO2 at 37uC. Calvarial osteoblastic cells: Calvarial bones were dissected from newborn osteoblast-specific CREB knockout mice and their control littermates. CREB knockout mice, which exhibit low bone mass [35,36], were generated by crossing CREBfloxed mice [55] with type I collagen I-a´1 Cre (Col1a1-2.3-Cre) mice. These mice were housed following standard LAR mouse housing protocol. All the mouse manipulations were performed under a Tulane IACUC approved protocol (#4196). Calvarial tissues were digested with 0.05% trypsin and 1.5 U/ml collagenase at 37uC on a rocking platform. Cell fractions 2 to 4 were collected and cultured in aMEM. CREB phosphorylation assay C2C12 cells cultured in 6-well plates were incubated with PTH, IBMX or KT5720 at different doses for 0 to 4 hours. Cells were lysed with SDS-RIPA buffer containing protease inhibitors PMSF (1 mM), aprotinin (10 mg/ml) and leupeptin (10 mg/ml). Samples were loaded on SDS-PAGE (Mini-Protein II Ready Gels, Bio-Rad, Hercules, CA), and proteins were electrophoretically separated under reducing conditions and then transferred onto PVDF membranes (Bio-Rad) in transblotting buffer (25 mM Tris, 192 mM glycine and 20% [v/v] methanol, pH 8.3) at 4uC for 1 hour. After blocking with 5% milk in TBS-T (0.1% Tween 20) for 1 hour at room temperature, membranes were incubated with antibodies against phosphorylated-CREB (Phospho-CREB, Ser133) or non-phosphorylated CREB (Cell Signaling Technology Inc. Danvers, MA) in 5% milk in TBS-T at 4uC overnight. After washes, membranes were incubated with horse-radish peroxidase (HRP)-conjugated secondary antibody (Amersham Biosciences, Buckinghamshire, UK) at 1:5,000 dilution at room temperature for 1 hour, then washed 6 times with TBS-T buffer for 5 minutes each. Immunoblots were detected using an enhanced chemilumi- nesence ECL system (Amersham). The antibodies on the same PVDF membranes were removed with stripping buffer and re- blotted with anti-b-actin antibody to detect b-actin levels for protein normalization. Alkaline phosphatase (ALP) activity assay All the tested osteoblast cells were seeded into 48-well plates. To test the effects of PTH, C2C12 cells were incubated with PTH at different doses for 24 hours. To test the effects of CREB overexpression, C2C12 and 2T3 cells were transiently transfected with CREB expression vector or empty vector using LipofectA- mine Plus reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. The transfected cells were cultured in the presence or absence of noggin at 500 ng/ml for 48 hours. To test the effects of CREB knockout, primary calvarial cells were harvested and cultured for 24 hours. All these cells were cultured in media supplemented with 2.5% FCS. Then, cells were lysed in 0.05% Triton X-100 buffer. ALP activity in cell lysates was spectrophotometrically quantitated at 405 nm using Sigma ALP reagents (Sigma-Aldrich, St. Louis, MO) and normalized with total cellular protein. CREB Activates BMP2 Expression in Osteoblasts Mineralized matrix formation Osteoblastic 2T3 cells were transfected with CREB expression vector or empty vector in 24-well plates and cultured with aMEM with 10% FCS. At confluence, the culture medium was supplemented with 5% FCS, ascorbic acid (100 mg/ml), and b-glycerol phosphate (5 mM). The medium was refreshed every other day. At days 14 and 21, the cultures were terminated and fixed in phosphate-buffered formalin for 10 minutes followed by von Kossa staining with 2% silver nitrate solution, and exposed to sunlight for 20 minutes. The area of mineralized matrix was stained black in the wells. Real time PCR Total RNA was purified from osteoblast cells in 6-well plates with various treatments, and reverse transcribed into cDNA. Quantitative PCR of mouse BMP2 mRNA was performed using a cDNA template and mouse BMP2 primers/probe, Mm01340178_m1 (Applied Biosystems, Carlsbad, CA) on 7300 Real Time PCR System (Applied Biosystems). The endogenous control was GAPDH detected using a VIC/MGB Probe (4352339E, Applied Biosystems). mRNA levels of other osteoblast marker genes including Runx2, Col1a1 and OCN were also quantitated by real time PCR using primers Mm00501578_m1, Mm00801666_g1*, Mm03413826_mH (Ap- plied Biosystems) respectively, with 18s rRNA for normalization. Promoter reporter assay C2C12 cells in 24-well plates were transfected with 0.5 mg of CREB responsive reporter CRE-Luc, or mouse BMP2 promoter luciferase reporter 22712/+165-Luc, or its truncated forms, or mouse BMP4 promoter reporter BMP4-Luc, and 0.1 mg of pSV- b-Galactosidase (b-Gal) expression vector (Promega, Madison, WI), using the LipofectAmine Plus Reagent (Invitrogen). Cells transfected with the BMP2 or BMP4 promoter reporters were also co-transfected with 0.2 mg of CREB or ATF4 expression constructs. The cells were cultured in the presence or absence of experimental compounds including PTH, IBMX or KT5720 for 24–48 hours and then lysed with Reporter Lysis Buffer (Promega). Luciferase activities of cell lysates were measured by a luciferase reporter assay kit (Promega) and normalized to b-Gal activity. RNA interference C2C12, 2T3 and UMR106 cells cultured in 6-well plates were transiently transfected with CREB siRNA or negative control siRNA (CREB ShortCutH siRNA Mix, New England Biolabs) using siRNA transfection reagents. Following confirmation of CREB protein levels in the cell lysates by Western blot at 24 to 48 hours after transfection (data not shown), BMP2 mRNA levels were determined as described above. Mutation of promoter binding sites The putative CREs in the BMP2 promoter reporter construct 2150/+165-Luc were mutated by deleting or replacing core nucleotides using GeneEditorTM in Vitro Site-Directed Mutagenesis System (Promega) following the manufacturer’s protocol. The phos- phorylated mutant primers for individual mutation of CRE1, CRE2 and CRE3 were p-tcgcggcccagctagcagaacgtccgtc, p-ctggtccctgagg- ccggatccagc agcagccttgcc, and p-cctgctcgaggctgtggatccagcacttggctg- gag, respectively. The luciferase activity of mutant reporters responding to CREB transfection were measured as described above. Chromatin immunoprecipitation (ChIP) C2C12 cells cultured in 100 mm Petri dishes were incubated with PTH at 50 nM for 6 hours. The cells were fixed with 1% PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e20780