Cell Culture and Transfection
C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium and 2T3 cells were cultured in α-minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum. Deletion constructs of the Smad6 promoter were co-transfected with Runx2, Smad1, Smurf1, or control expression plasmids into C2C12 and 2T3 cells and treated with BMP-2 (100 ng/ml) or control medium in 6-cm culture dishes using the Lipofectamine 2000 reagents (Invitrogen). The total amount of DNA transfected was constant in the experiments. Luciferase assays were performed 24 h after transfection, and chromatin immunoprecipitation (ChIP) assays were performed 2 h after BMP-2 treatment.
Real-time RT-PCR Analysis
C2C12 and 2T3 cells were treated with different concentrations of BMP-2 (50, 100, and 200 ng/ml) or transfected with different amounts of Runx2 or Smurf1 expression plasmids (0.2, 0.4, and 0.8 µg/dish, 6-mm culture dish). Total RNA of cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. After reverse transcription reaction, real-time PCR was performed by an ABI PRISM 7000 sequence detection system according to the manufacturer’s instructions. The reaction mixture contained 1×EvaGreen dye, 0.5 pm of each primer, 2.5 mm MgCl2, and 0.5 µl of cDNA from 20 µl of reverse transcription reaction. The conditions of real-time PCR were as follows: 94 °C for 4 min followed by 40 cycles at 94 °C for 30 s, 60 °C for 1.5 min. There is no nonspecific amplification determined by dissolve curve. There are four samples in each group. The real-time RT-PCR results were reported as the fold induction of relative light units for the treatment over vehicle after normalization to GAPDH expression. The primers used for this analysis were: Smad6: 5′-TACCACTTCAGCCGGCTCTG-3′ (forward) and 5′-AGTACGCCACGCTGCACCAGT-3′ (reverse); GAPDH: 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTTGCTGTA-3′ (reverse).
Western Blot Analysis
C2C12 and 2T3 cells were treated with different concentration of BMP-2 (50, 100, and 200 ng/ml) or transfected with different amounts of Runx2 or Smurf1 expression plasmids (0.2, 0.4, and 0.8 µg/dish, 6-mm culture dish). Cells were lysed on ice for 30 min in a buffer containing 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin) and phosphatase inhibitors (1 mm NaF and 1 mm Na3 VO4). Proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using the anti-Smad6 (Imgenex, San Diego, CA) and anti-β-actin (Sigma) antibodies. Immunostaining was detected using an enhanced chemiluminescence (ECL) system (Amersham Biosciences).
C2C12 and 2T3 cells were co-transfected with wild-type (WT) or mutant deletion constructs of the Smad6 promoter (−2006/+45, −1191/+45, −829/+45, and −374/+45) and Runx2, Smad1, Smurf1, mutant Smurf1 (C710A), or control expression plasmids and treated with BMP-2 (100 ng/ml) or control medium. Cell lysates were extracted 24 h after transfection, and luciferase activity was measured using a Promega dual luciferase reporter assay kit (Promega, Madison, WI).
PCR-based Site-directed Mutagenesis
Smad6 promoter constructs, −2006/+45 and −1191/+45 with mutations at either OSE2-a or OSE2-b site were constructed using Stratagene QuikChange Site-directed mutagenesis kit and cloned into pGL3 vector (Stratagene). The mutations were confirmed by sequencing the entire promoter fragment.
Electrophoresis Mobility Shift Assay
Nuclear extracts (5 µg) from C2C12 cells were incubated with radioactive end-labeled double-stranded oligonucleotide probes containing consensus binding sites for Runx2 in the mouse Smad6 promoter (5′-CCGGGTCGCGGTGGGTTCACC-3′, the binding site is highlighted). Incubation was performed in 10 µl of binding buffer (10 mm Tris-HCl, pH 7.5, 150 mm KCl, 1 mm EDTA, 0.1% Triton-X 100, 10% glycerol, 1 µg of poly(dI-dC), 1 mm dithiothreitol), for 15–20 min at room temperature. For competition, the cold wild type or mutant oligonucleotides (5′-CCGGGTCGCGGTGTTTTCACC-3′ for the mutant oligo, nucleotide mutations indicated by underscore) were added in a 1:1 or 1:20 excess. For supershift assay, the nuclear extract was incubated with Runx-2 specific (Abcam, Cambridge, MA) or control antibodies for 30 min before addition of the labeled probe. After incubation with labeled probe for an additional 20–30 min, samples were fractionated on a 4% native polyacrylamide gel and visualized by exposing dry gel to Kodak Film.
ChIP assay was performed using a kit from Upstate Technologies (Lake Placid, NY) with the following modifications. C2C12 or 2T3 cells were treated with 1% formaldehyde at 37 °C in the presence of 4% CO2 for 15 min. Cells were sonicated on ice for 6 times at 50 watts using a Sonic Dismembrator (Scientz, China) for 25 s with a 60-s interval between each sonication. The following antibodies (10 µg) were used: anti-Runx2 antibody (MBL), anti-Myc antibody (Santa Cruz Biotechnology), and anti-FLAG antibody (Sigma) with 100 µg of chromatin per ChIP. The amounts of each specific DNA fragment in immunoprecipitates were determined by PCR or quantitative PCR reactions. The primers used for this analysis were: A (−1108 to −937) 5′-GTGCTATGCGCGTTTGCAAGT-3′ (forward) and 5′-CTCAAGCTCACGTCGTGCCTT-3′ (reverse); B (−1968/−1839) 5′-CAGTCCAGAGCACAGGTATCCT-3′ (forward) and 5′-CCGAGCTCAATGAAAGAAATTCC-3′ (reverse); C (+281/+432) 5′-GCGCCAAAGGGTATCGTATG-3′ (forward) and 5′-AGGCTCAGCTCGGCTGCCCA-3′ (reverse). The conditions of PCR for the detection of each specific DNA fragment were as follows: 94 °C for 4 min followed by 28 cycles at 94 °C for 30 s, 62 °C for 30 s, 72 °C for 60 s.
For the quantitative ChIP (qChIP), a standard curve was generated using primer set A. Copy numbers for the DNA fragment −1108 to −937 in each anti-Runx2-and anti-Smurf1-immunoprecipitated samples were determined and compared with copy numbers of the DNA fragment without IP (input DNA). Anti-FLAG antibody was used as control for IP. The percentage of the input was then calculated. The final value was the percentage input obtained with specific antibody minus the percentage input obtained with the anti-FLAG control antibody. The dissociation curve was determined for each quantitative PCR reaction to assure that a single band was produced. Each data point represents four independent samples.