bones using an Isomet saw with a diamond wafer blade for cortical studies. The sections were polished and ground to a thickness of approximately five microns with a glass plate coated in aluminum oxide powder, mounted on a microscope slide, and examined with a fluorescent microscope. For trabecular studies, distal femurs were dehydrated, embedded in methylmethacrylate and cut longitudinally into five micron-thick sections. The dynamic properties of trabecular bone surfaces were assessed on unstained sections at 200X using the OsteoMetricsTM system by measuring the distance between the two calcein labels, while the properties of the cortical periosteal and endosteal surfaces were determined by measuring the distance between the tetracycline label and second calcein label as previously described [59]. For static measures, the distal femurs were embedded in methylmethacrylate and cut longitudinally into five micron-thick serial sections. Serial sections were stained with Goldner’s trichrome or von Kossa to detect mineralized bone, osteoid, cells and adipocytes; toluidine blue to detect osteoblasts; or TRAP (to distinguish osteoclasts) for static histomorphometric measures. Data were collected at 2006 magnification using OsteoMeasure software. Data from Hdac3 mutant mice were compared to those from wildtype mice with student t-tests. Growth Plate Analyses Femur lengths were measured from the inter-condylar notch to trochanteric notch using a caliper. The distal femurs of 12-5.5 week-old male mice were embedded in methylmethacrylate and cut longitudinally into five micron-thick serial sections. Serial sections were stained with Goldner’s trichrome. A technician blind to sample identities measured growth plate depth (mm) and hypertrophic zone depth (mm) with a previously published technique that was adapted for the KS300 computerized image analysis system (Carl Zeiss Vision GmbH, Hallbergmoos, Germany) [60,61]. Briefly, two sections were obtained from each tissue sample and scanned at the growth plate area using the Zeiss AuxioCam MRc at the same magnification with the calcification zone horizontal to the bottom edge of the scan’s frame of reference. Each growth plate or hypertrophic zone was outlined by hand. Average thickness was determined by comparing thicknesses in the outlined area from top to bottom. Values obtained from sections made from the same tissue sample were then averaged to give overall mean values for cambium thickness and hypertrophic zone thickness. Hypertrophic zone thickness was then normalized to growth plate thickness in order to yield percentage hypertrophic zone in growth plate. Statistical analysis was done using a student’s t test and Wilcoxon Rank Sum non-parametric where appropriate. Statistics are reported with standard deviations. Calvarial Explants To enrich for cellular protein and obtain RNA from bone tissue, calvarial bones from four to five day-old pups were dissected from soft tissues, rinsed with HBSS, and digested in collagenase digestion medium [52] on an orbital shaker for 20 minutes at 37 degrees at 125 rpm. Following digestion, explants were washed once with PBS and snap frozen in liquid nitrogen. The remaining bone tissues were crushed under liquid nitrogen with a mortar and pestle and then resuspended in either TRIzol (Invitrogen) for RNA extraction or modified-RIPA buffer supplemented with protease inhibitors for protein isolation. Microarray studies RNA from calvarial explants was purified using TRIzol according to the manufacturer’s protocol (Invitrogen) and reverse transcribed using Qiagen’s Quantitect Reverse Transcription Kit. Hdac3 Regulates Bone Density Microarray experiments were performed in the Mayo Clinic Advanced Genomic Technology Center using the Illumina MouseRef-8 BeadChip array. Two independent experiments were performed; each experiment used RNA from four wildtype and four CKO litter-matched mouse calvaria. The raw data from eight samples in each experiment were pre-processed using Beadstudio (Illumina Inc, San Diego, CA). Raw data were submitted to the MIAME-compliant GEO database (Accession number: GSE21087). Genes that were not expressed in eight calvarial RNA samples (detection p$0.05) were eliminated from analysis, leaving 10,500 gene transcripts out of 19,000 for the final analysis. A paired t-test was used to compare differential gene expression between the wildtype and CKO groups. Genes with fold changes greater than 1 standard deviation in both experiments were used for pathway analysis using MetaCore software (GeneGo Inc, St. Joseph, MI, USA). Genes that were significantly (1.4 fold, p#0.05) differentially expressed between the WT and CKO groups were subjected to reverse transcription (RT) and real-time semi- quantitative (q) PCR analysis for confirmation. RT-qPCR To verify microarray results, calvarial RNA samples from litter- matched wildtype and CKO mice were reverse-transcribed with Quantitect Reverse Transcription Kit (Qiagen). Gene specific primers sequences are available upon request. Real time qPCRs were performed using 15 ng of cDNA per 10 ml using Bio-Rad iQ SYBR Green Supermix in the Eppendorf epMotion 5070 and the Applied Biosystems 7900 HT Fast Real Time PCR System. Transcript levels were normalized to three housekeeping genes (YWHAZ, GAPDH, and HRPT1), which were our most stable osteoblast genes as determined using GeNorm software [62]. Quantification was done using the 22DDCt method [63]. Western blotting Protein extracts from calvarial explants were sonicated and resolved by SDS-PAGE. Immunoblotting was performed with antibodies recognizing Hdac3 (1:1000, Abcam, Ab63353). Luciferase Assays p21 reporter assays were performed as previously described [25]. Supporting Information Figure S1 Photograph of representative 5.5. week-old mice used in this study. The animals’ weights are listed below the photos. Found at: doi:10.1371/journal.pone.0011492.s001 (0.57 MB TIF) Figure S2 MicroCT Reconstructions of skulls from 5.5 week-old male Hdac3 WT, Het and CKO mice. These are alternative angles of the microCT reconstructions shown in Figure 1E. Found at: doi:10.1371/journal.pone.0011492.s002 (0.96 MB TIF) Figure S3 Hdac3-Depletion in Osx-expressing cells decreases trabecular bone volume in distal femora. A-E. Structural parameters of distal femurs from microCT reconstruction of 12 week-old male mice. n = 4-5 per group, *p,0.05. Found at: doi:10.1371/journal.pone.0011492.s003 (0.10 MB TIF) Figure S4 HDAC3 represses the p21(CIP1) promoter. C2C12 cells were transfected with a p21-luciferase reporter and increasing concentrations of pCMV-HDAC3. Firefly luciferase activity was normalized to renilla-luciferase activity to control for transfection efficiency. Data are shown relative to cells transfected with an empty CMV vector. Found at: doi:10.1371/journal.pone.0011492.s004 (0.04 MB TIF) PLoS ONE | www.plosone.org 13 July 2010 | Volume 5 | Issue 7 | e11492