related diseases such as Ehlers-Danlos syndrome. In this regard, considering the capability of Prdm5 to bind genomic loci encoding multiple collagen molecules, it may be that Prdm5 affects the levels of specific collagen molecules in a context-specific fashion. These features support the role for Prdm5 as an important regulator of extracellular matrix genes transcription during the process of bone formation, and extend the need for studies characterizing this protein in different clinical settings. Materials and Methods Animal experiments To generate the Prdm5LacZ strain, ES cell clone AV0702 from the Sanger Institute Gene Trap project was microinjected into C57BL/6 blastocysts. The integration site of the b-geo cassette was established using long range PCR with primers spanning different regions of intron 2 and a genotyping strategy was devised accordingly. All experiments were performed on mice backcrossed for at least 6 generations into the C57BL/6 strain and comparisons between WT and mutant animals always refer to littermates. All animal experimentation was performed with approval from Danish authorities (Dyreforsøgstilsynet, protocol number 2006/562-43) and the Regierung von Oberbayern (Government of Upper Bavaria). Histological staining and immunohistochemistry For whole mount X-gal staining, embryos were deskinned (after E15) and fixed in 0.25% glutaraldehyde (Sigma). Stainings were performed by incubation in staining solution containing 0.02% NP40, 0.01% Sodium Deoxycholic acid, 2 mM MgCl2, 20 mM Tris-HCl (pH 7.4), 50 mM K-Ferrocyanide, 50 mM K-Ferricya- nide, 1 mg/mL X-gal in PBS (chemicals purchased from Sigma). Stained embryos were post-fixed in 4% paraformaldehyde and cleared in increasing concentrations of glycerol in 1% KOH. Images were acquired with a Leica D-Lux 3 on Leica S6D stereoscope. Von Kossa staining was performed by staining dehydrated sections in 1% AgNO3 under UV light, followed by incubation with 5% Sodium thiosulfate. Picrosirius red stainings were performed as described [37]. Images were acquired under polarized light using a BX51 Olympus microscope. In-situ hybridization was performed as previously described [38] and protocols for immunofluorescence stainings are described in Text S1. Cell culture and transfections Primary human osteoblasts (kindly provided by Bente Langdahl, A˚rhus University, Denmark) were isolated from bone biopsies of healthy donors. MC3T3 cells were maintained in Alpha-MEM (Gibco) with 10% FBS (Hyclone) and 1% penicillin-streptomycin (Gibco). ATDC5 cells were maintained in DMEM:F12 (Gibco) media supplemented with 5% FBS, 10 mg/ml human transferrin (Roche) and 30 nM sodium selenite (Sigma). Calvarial osteoblasts were derived as previously described [21]. Differentiation proto- cols and transfection/transduction techniques are detailed in Text S1. Co-immunoprecipitation, immunoblotting, and gene expression analyses For co-immunoprecipitation experiments HEK293 cells were transfected with indicated plasmids and, 48 hours after, lysed in ELB buffer (150 mM NaCl, 50 mM HEPES, 0.1% Igepal) and incubated with HA-agarose beads (Sigma) overnight. After washes, proteins were recovered by boiling beads in SDS Laemmli buffer. Prdm5 Promotes the Transcription of ECM Genes For whole cells lysate immunoblotting, cells were harvested and lysed in RIPA buffer and subjected to standard SDS-PAGE. Protocols and antibodies used are detailed in Text S1. Total RNA was extracted from cell pellets using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instruc- tions. cDNA was synthetized using TaqMan Reverse Trancription Reagents (Applied Biosystems). qRT-PCR was performed with the One Step plus Sequence Detection System (Applied Biosystems) using Fast SYBR green master mix reagent (Applied Biosystems). Gene expression levels were normalized to the average of at least two housekeeping genes. qPCR primers sequences are listed in Table S3. For MC3T3 microarray data, RNA was amplified and labeled using TotalPrep RNA Amplification Kit (Ambion) and hybridized to MouseWG-6 v2.0 Expression BeadChip array (Illumina) according to manufacturer’s recommendation. Chromatin immunoprecipitation and Deep sequencing Chromatin immunoprecipitation assay (ChIP) protocol and antibodies used are detailed in Text S1. Libraries for sequencing were obtained using the ChIP-seq DNA sample prep kit (Illumina) according to manufacturer recommendations and samples were sequenced on a Genome Analyzer II sequencer (Illumina). Primers for ChIP-qPCR experiments are listed in Table S3. Bioinformatic analysis Microarray and ChIP-seq analyses are detailed in Text S1. mCT and Peripheral quantitative computed tomography E18.5 embryos were scanned using a micro CT scanner (model 1172, Skyscan, Belgium) at 50 kV, 200 mA and a 0.5 mm aluminium filter. Adult mice at 5 weeks of age were analyzed by pQCT using Stratec XCT Research SA+ (Stratec Medizintechnik GmbH, Germany) at the German Mouse Clinic [39]. Analyses details are provided in Text S1. Supporting Information Figure S1 Prdm5LacZ/LacZ mice do not display gross abnormalities and PRDM5 is expressed in osteoblasts. A) Left panel. Western blot of +/+ and LacZ/LacZ mouse embryo fibroblasts using serum from mice immunized with recombinant Prdm51-142. Right panel. qPCR for Prdm5 wt and Prdm5-bgeo fusion alleles in mouse embryo fibroblasts of the three genotypes. B) qPCR for Prdm5 wild type transcript in a panel of tissues from wild type and Prdm5LacZ/ LacZ mice. Results are shown as the average of 3 animals per group 6 standard deviations. C) Number of mice obtained at weaning with different genotypes from Prdm5+/LacZ intercrosses. In parenthesis the expected numbers according to Mendelian ratios. Statistical differences were calculated by Chi-square test. D) Weight of wild type and Prdm5LacZ/LacZ mice up to 56 weeks of age. E) qPCR analysis for PRDM5 in primary human osteoblasts from 6 different donors (black bars) and a panel of human immortalized cell lines of different origins (grey bars). F) Western blot analysis of Prdm5 protein expression in the indicated mouse cell lines. * = unspecific bands for equal loading. (PDF) Figure S2 Prdm5 knockdown affects osteogenic differentiation in vitro, but not chondrogenic differentiation. A) Left panel. qPCR of Prdm5 levels in chondrogenic ATDC5 cells infected with lentiviral shRNA constructs against the Prdm5 transcript (shPrdm5-a) and control construct. Right panel. Western blot measuring Prdm5 protein levels in the same cells. Actin is used as a control for equal protein loading. B) Upper panel. Quantification PLoS Genetics | www.plosgenetics.org 11 May 2012 | Volume 8 | Issue 5 | e1002711