detection and significance were ,0.05. Since the variation between the replicates of the treatment DMSO+BMP-2 was high (correlation coefficient of 0.8351), only one of the replicates was considered for further analysis. The excluded sample was not considered for the significance test, although the detection P-values and fold change were used for filtrations. A cut-off of .1.4 fold change with respect to DMSO control treatment was set to extract differentially expressed genes in group-wise comparisons. A Venn diagram was generated from these regulated lists employing the VENNY interactive tool (http://bioinfogp.cnb.csic.es/tools/venny/index.html) and heat maps were generated using the TIGR-MEV software [60,61]. All microarray data presented in this study is MIAME compliant and has been submitted to GEO database (accession number GSE29373). Functional annotation and pathway analysis were done using the DAVID platform version 6.7 with default parameter settings [63,64]. However, a subset of the regulated genes from each comparison was validated by quantitative real-time PCR (qPCR), to ascertain the reliability of the data. This was performed with cDNA generated from RNA used for whole genome analysis as well as with RNA from two independent experiments. qPCR analysis To analyze mRNA expression after 6 h of BMP-2 stimulation (Figure 4A B), C2C12 cells were treated like described above in microarray analysis. To analyze the effect of inhibition of endocytosis by dynasore on mRNA expression after initiation of osteoblast differentiation (Figure 5C–G), cells were treated as described below for ALP assay. Total RNA was isolated according to manufacturer’s instructions using NucleoSpin RNA II Isolation Kit (Machery-Nagel). Complementary DNA (cDNA) was synthesized from 0,5–1,5 mg RNA and used in SYBRGreen qPCR. The amount of investigated transcript was determined relative to the housekeeping genes hypoxanthine- phosphoribosyltransferase (HPRT) or glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH). The sequences of mouse specific primers for qPCR analysis are shown in Table S1. All measurements were done in duplicates (StepOnePlus Real-Time PCR System, Applied Biosystems) and C(T) values were determined with StepOne Software (Applied Biosystems). Mean normalized expression (MNE) and the corresponding standard error were calculated according to a previous publication [62]. Alkaline phosphatase assay C2C12 cells were seeded at high density to reach confluency after 24 h. Cells were washed with PBS, starved for 2 h in serum free medium and subsequently stimulated with 5 nM, 30 nM and 60 nM BMP-2 in DMEM supplemented with 2% FCS, 1 mM Glutamine, Penicillin (100 units/ml) and Streptomycin (10 mg/ml) for 72 h. To study the effect of endocytosis inhibition on osteoblast differentiation by measuring ALP activity (Figure 5A), 40 mM dynasore or 0,05% DMSO were included during starvation and initial 4 h of stimulation with BMP-2 in serum free medium, then stimulation medium was replaced by medium containing BMP-2 only. To study the importance of the initial phase of BMP-2 stimulation on ALP activity (Figure 5B), BMP-2 in the indicated concentration was only added for 4 h and afterwards the cells were incubated in medium without BMP-2 for additional 68 h. Cells were lysed by incubation for 1 h in 100 ml ALP 1 buffer (0.1 M glycine pH 9.6, 1 mM MgCl2, 1 mM ZnCl2, 1% v/v NONIDET P-40). After addition of 100 ml ALP 2 buffer (0.1 M glycine References Spatial Segregation of BMP/Smad Signaling pH 9.6,1 mM MgCl2,1 mM ZnCl2, 2 mg/ml para-nitrophenyl- phosphate (pNPP) (Roth)) ALP enzymatic activity was measured using a microplate reader (Tecan) at 405 nm. The results are represented as mean 6 s.d. of triplicates. Statistical analysis Results are presented as mean 6 standard deviation (s.d.) or standard error (s.e.). Statistical significances (P-values) were calculated by two-tailed Student’s t test. P-values of P,0.05 were considered to indicate statistical significance. Supporting Information Figure S1 Linear correlation coefficients of biological duplicates subjected to whole genome expression anal- ysis. (TIF) List of mouse primers used for qPCR Table S1 analysis. (DOC) Table S2 Complete lists of genes regulated and signif- icantly detected in microarray analysis (Figure 3A). Worksheets: 1) genes exclusively regulated by BMP-2+DMSO (254 genes), 2) genes exclusively regulated by BMP-2+Dynasore (30 genes), 3) genes exclusively regulated by Dynasore (39 genes), 4) genes commonly regulated by BMP-2+DMSO or BMP- 2+Dynasore (93 genes), 5) genes commonly regulated by BMP- 2+DMSO or by Dynasore (71 genes), 6) genes commonly regulated by BMP-2+Dynasore or by Dynasore (610 genes), 7) genes commonly regulated in all treatments (1117 genes). Expression signals relative to DMSO are displayed in log2 scale, detection P-values of all treatments are included. (XLS) Table S3 List of BMP-2 induced genes which are depen- dent on dynamin-dependent endocytosis (Figure 3B) (483 genes). Expression signals relative to DMSO are displayed in log2 scale, detection P-values of all treatments are included. (XLSX) Table S4 List of BMP-2 induced genes which are inde- pendent of dynamin-dependent endocytosis (Figure 3C) (20 genes). Expression signals relative to DMSO are displayed in log2 scale, detection P-values of all treatments are included. (XLSX) Acknowledgments We thank Stefan Mundlos, Richard Reinhardt and Aydah Sabah (Max Planck Institute for Molecular Genetics, Germany) for support in microarray performance, Robert Benezra (Memorial Sloan Kettering Cancer Center, USA) and Yoshifumi Yokota (University of Fukui, Japan) for the gifts of Id1 and Id2 reporter constructs, respectively, and Isabel Casanova (Freie Universitaet Berlin, Germany) for technical assistance. Author Contributions Conceived and designed the experiments: PK. Performed the experiments: EH RB PP. Analyzed the data: EH RB PP YIH PK. Contributed reagents/materials/analysis tools: PK. Wrote the paper: EH RB YIH PK. 1. Kishigami S, Mishina Y (2005) BMP signaling and early embryonic patterning. Cytokine Growth Factor Rev 16: 265–278. 2. Sieber C, Kopf J, Hiepen C, Knaus P (2009) Recent advances in BMP receptor signaling. Cytokine Growth Factor Rev 20: 343–355. 3. Urist MR (1965) Bone: formation by autoinduction. Science 150: 893–899. 4. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, et al. (1988) Novel regulators of bone formation: molecular clones and activities. Science 242: 1528–1534. PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e25163