Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions and assayed 24 h after transfection. The ligand BMP-2 was generously provided by W. Sebald (University of Wuerzburg, Wuerzburg, Germany). Human transferrin labelled with AlexaFluor594 (Invitrogen) was stored light protected at 4uC as a stock solution of 5 mg/ml containing 2 mM sodium azide according to manufacturer’s instructions. The endocytosis inhib- itor dynasore (Sigma) was aliquoted under argon as a stock solution of 80 mM in DMSO and kept light protected at 280uC [58]. The following antibodies were used: anti-pSmad1/5/8 (#9511, Cell Signaling), anti-GAPDH (#2118, Cell Signaling), anti-Smad1 (sc-7965, Santa Cruz Biotechnology) and anti-Histon H3 (#9715, Cell Signaling). Inhibition of dynamin-dependent endocytosis To inhibit endocytosis in signaling assays, serum starved C2C12 cells were washed with PBS and pre-treated with 40 mM dynasore or 0,05% DMSO as control in DMEM supplemented with 0% FCS, 1 mM Glutamine, Penicillin (100 units/ml) and Streptomy- cin (10 mg/ml) (serum free medium) for 30 min up to 2 h. During stimulation treatments, dynasore and DMSO were added to the indicated medium if not stated otherwise. Transferrin uptake To assess endocytosis inhibition efficiency of dynasore, C2C12 cells were subjected to transferrin uptake [14]. Cells were grown on glass coverslips and pre-treated with 40 mM dynasore or 0,05% DMSO in serum free medium in parallel with the respective experiment. The uptake experiment was performed by incubating the cells in 30 mg/ml Alexa594-transferrin in Opti-MEM (Invitro- gen) including dynasore or DMSO for 15 min at 37uC. Cells were put on 4uC and washed 3 times with ice-cold PBS/10 mM MgCl2 before fixation with 4% PFA for 20 min at room temperature. Cells were washed two times with PBS and incubated for 3 min at 220uC with methanol. After 5 washes with PBS, cells were stained with 1 mg/ml Hoechst dye in PBS for 2 min at room temperature and mounted in FluoromountG (Southern Biotech). Cells were imaged with 63-fold magnification with an Axiovert 200 M fluorescence microscope (Zeiss) by using the same exposure time for all slides. Pictures were analyzed using SlideBook Software (Intelligent Imaging Innovations, Inc.) by quantifying the total amount of Alexa594- transferrin accumulated within cell boundaries. Transferrin uptake is represented in arbitrary units (AU) as mean 6 s.d. of fluorescence intensity inside of a minimum of 400 cells for each condition. Smad phosphorylation assay and Cytosplasmic-Nuclear fractionation assay C2C12 cells were starved for 22 h in DMEM supplemented with 0,5% FCS, 1 mM Glutamine, Penicillin (100 units/ml) and Streptomycin (10 mg/ml), then washed once with PBS and incubated with serum free medium including 40 mM dynasore or 0,05% DMSO for 2 h. Cells were subsequently stimulated with 10 nM BMP-2 in serum free medium containing dynasore or DMSO for the indicated time periods. To analyze the Smad phosphorylation status, cells were directly lysed in 16 Laemmli- buffer. To analyze the subcellular localization of Smad proteins by fractionation, cells were prepared according to manufacturer’s instructions using ProteoJet Cytoplasmic and Nuclear Protein Extraction Kit (Fermentas). Samples were subjected to SDS- PAGE and transferred to nitrocellulose membranes by Western blotting. The membranes were probed with antibodies specific for C-terminal phosphorylated Smad1/5/8 and GAPDH or Histone H3 as loading control or control for fractionation, respectively. Spatial Segregation of BMP/Smad Signaling Immunofluorescence microscopy To analyze subcellular localization of Smad1 protein by immunofluorescence, C2C12 cells were grown on glass coverslips and starved in serum free medium for 1,5 h. Cells were washed with PBS and pre-treated for 30 min in serum free medium containing 40 mM dynasore or 0,05% DMSO prior to stimulation with 10 nM BMP-2 for 30 min in serum free medium containing dynasore or DMSO. Subsequently, cells were fixed in 4% PFA for 20 min at room temperature and probed with anti-Smad1 antibody and an AlexaFluor594 goat anti mouse antibody (Invitrogen) [59]. Cells were stained with 1 mg/ml Hoechst dye in PBS for 2 min at room temperature and mounted in FluoromountG (Southern Biotech). Cells were analyzed with 63-fold magnification using an Axiovert 200 M fluorescence microscope. Quantification of relative fluores- cence in the nucleus was assessed using SlideBook Software (Intelligent Imaging Innovations, Inc.) to determine the ratio of fluorescence intensity of Smad1 localization in the nucleus versus Smad1 localization in the cytoplasm of a minimum of 100 cells. The results are represented in arbitrary units (AU) as mean 6 s.d. Reportergene Assay To analyze BMP-2-dependent transcriptional responses, C2C12 cells were transfected with a BMP response element reportergene construct (BRE-Luc) [15], a reporter construct containing the promoter regions of Id1 ((21575)-(+88), Id1-Luc) [17] or Id2 ((24516)-(+80), Id2-Luc) [18] together with a constitutively active renilla luciferase (RL-TK) (Promega) as internal control. After 24 h, cells were starved for 3 h in serum free medium, washed with PBS and pre-treated for 1 h in serum free medium containing 40 mM dynasore or 0,05% DMSO prior to stimulation with 3 nM BMP-2 for 6 h or 24 h. During stimulation for 24 h, 0,5% FCS was added to the medium. Cell lysis and luciferase measurements were carried out according to manufacturer’s instructions (Dual- Luciferase Reporter Assay System, Promega) and measured using a Mithras LB 940 luminometer (Berthold Detection Systems). The ratio of BMP-2 induced luciferase and renilla luciferase activity is shown as relative luciferase activity (RLA). The results are represented as mean 6 s.d. of triplicates. Illumina BeadChip hybridization and data analysis C2C12 cells were starved for 2 h in serum free medium, washed with PBS and pre-treated for 2 h in serum free medium containing 40 mM dynasore or 0,05% DMSO and then stimulated with 30 nM BMP-2 in serum free medium containing dynasore or DMSO for 6 h. Samples were subjected to gene expression analysis as biological duplicates. Whole genome mRNA expression analysis was performed using MouseRef-8 v2.0 Expression BeadChips (BD-202-0202, Illumina). Total RNA was isolated using NucleoSpin RNA II Isolation Kit (Machery-Nagel) followed by quality assessment using both spectro- photometer (NanoDrop Technologies) and agarose gel electropho- resis. A total of 400 ng RNA was used as input for microarray experiments. Biotin-labelled cRNA was generated, employing a linear amplification kit (Ambion). Subsequently hybridization, washing, Cy3-streptavidin staining and scanning of hybridized chips was performed using Illumina Beadstation 500 platform (Illumina) according to manufacturer’s instructions. For gene expression data analysis, raw data were normalized using the rank invariant normalization algorithm included in BeadStudio 3.0 software (Illumina). To assess correlation coefficients, BeadStudio 3.0 software was utilized (Figure S1). For obtaining the list of significant differentially regulated genes, one-way ANOVA was performed using TIGR-MEV software [60,61]. A gene was considered to be significantly regulated, only if the corresponding P-values for both PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e25163