Following washes with 10%FBS/DMEM, cells were incubated with an anti-mouse secondary antibody conjugated to FITC (dilution 1:100 in 10%FBS/DMEM) for 30 minutes in the dark. After additional washes (2x) in 10%FBS/DMEM, cells were resuspended in 500 ml 10%FBS/DMEM and analyzed on the FACS instrument (Becton-Dickinson). The costaining with anti-BrdU-PE antibody was performed incubating the cells with bromodeoxiuridine (Roche) for 6 hr. After that, we proceeded with the anti-cTnI staining as previously described; then, cells were fixed with Cytofix-Perm solution (Becton Dickinson) for 209 on ice and treated with DNAseI (Quiagen) to expose the DNA (1 hr at 37uC). Treated cells were incubated with anti-BrdU-PE (BD) for 309 in the dark and analyzed on cytofluoremeter after extensive washes. The number of labeled cells were determined with a FACSCalibur and analyzed by FlowJo software. Immunofluorescence HEK293T cells were cultured on chamber slides (Lab-Tek II Chamber Slide w/Cover RS Glass Slide Sterile, Nunc) and transfected with the vectors pcDNA3X (+) HA-sik1 (1–249) and pcDNA3X (+) HA-rSik1 using the PolyFect Transfection Reagent (Qiagen). After 48 hours of transfection, cells were washed with PBS (Invitrogen), and fixed for 10 minutes in 4% paraformalde- hyde. After extensive washing in PBS, the fixed cells were incubated with 10% of Bovine Serum Albumin and 0.1% Tween 20 diluted in PBS (Invitrogen) for 1 hour at room temperature, and then incubated with an anti-HA antibody (dilution 1:250; Sigma-Aldrich) for 2 hours at room temperature. After extensive washing, the antigen-antibody complexes were visualized with anti-rabbit IgG-FITC conjugate IgG (dilution 1:100; Amersham Biosciences) for 1 hour at room temperature. Nuclei were counterstained with DAPI. EB-derived neurons were plated into gelatin-coated 100 mm dishes (Nunc), washed with PBS (Invitro- gen) and fixed for 10 minutes in 4% paraformaldehyde. The fixed EBs were incubated with 10% of Normal Goat Serum (Dako), 1% of Bovine Serum Albumine, 0.1% Triton X-100 diluted in PBS for 30 minutes at room temperature, and then reacted with an anti- bIII tubulin antibody (dilution 1:400; Sigma-Aldrich) for 2 hours at room temperature. The antigen-antibody complexes were visualized with rabbit anti-mouse IgG-FITC conjugate IgG (dilution 1:35; Dako) for 1 hour at room temperature. Nuclei were counterstained with DAPI. Images were taken through a digital camera (Zeiss Axiocam 130061030 pixel) mounted on an Axioplan 2 microscope (Carl Zeiss) with a 60x 1.40 lens (Carl Zeiss) at room temperature using AxioVision software (Carl Zeiss). For MF20 experiment, the fixation was performed with methanol: acetone (7:3). Immunoprecipitation and Kinase Assay Cells (26106) plated on a 10-cm dish were transformed with 6 mg of expression plasmids for FLAG-tagged wild type and mutant sik1 by using 20 ml of LipofectAMINE 2000 (Invitrogen). After 36 h of incubation, cells were lysed in 0.7 ml of lysis buffer. The FLAG-tagged sik proteins were immunoprecipitated with an anti- FLAG M2 affinity Gel (SIGMA). Aliquots of purified FLAG-SIK1 were then subjected to western blot analyses with the anti-FLAG-HRP antibodies and to in vitro kinase assays. Purified SIKs were incubated in 40 mM MOPS/NaOH pH 7.0, 1 mM EDTA with 0.1 mM of AMARA substrate (Enzo Life Sciences) in the presence of 25 mM MgAc and 0.5 mCi (18.5 kBq) of [c-32P]-ATP at 30uC for 30 min. The kinase reaction was stopped by adding 5 ml of 3% phosphoric acid. An aliquot was transferred to P81 filters (Whatman) and after Sik1 Controls Cardiomyogenesis extensive washes in 75 mM phosphoric acid read in a scintillation counter. RNA Preparation, RT-PCR and Q-PCR Total RNAs from either undifferentiated ES cells or differentiating EBs were extracted using RNeasy Miny Kit (Qiagen). First strand cDNA was synthesized from 1 mg of total RNA using QuantiTect Reverse Transcription kit (Qiagen). RT- PCR was performed on cDNA synthesized from wild-type and sik1flp/flp + HAsik1 ES cells using LA-TAQ (TAKARA). sik1UP and sik1LW primers were used to amplify the sik1 transcript and GAPDH as an internal control. Amplification products were analyzed on ethidium bromide-stained 1% agarose gels and visualized at Gel Doc 2000 (Bio-Rad) using Quantity One software. Q-PCR was performed on the Light Cycler 480 real- time PCR system (ROCHE) using SYBR Green (Light Cycler 480 SYBR Green I master, ROCHE). Each sample was amplified in triplicate. The primers used are available on request. Microarray Hybridization and Analysis Transcriptome analysis was performed comparing total RNA extracted from wild-type and mutant homozygous ES cells. The labeling of the RNAs, the hybridization on Affymetrix GeneChip MOE 430A 2.0 and analysis were performed by the Boston University Microarray Resource. The array data analysis was performed using the RMA algorithm. The array experiments were submitted to MIAMEXPRESS and the assigned accession number is E-MEXP-1646. Detection of Functional DNA Motifs The following bioinformatics approach was used to detect the presence of eventual functional DNA motifs regulating the expression of the genes found to be differentially expressed. Affymetrix probeset IDs were used to retrieve a list of corresponding Ensembl transcript IDs from the Ensembl database (release 56, based on Mus Musculus NCBIM37). Transcript identifiers were then used to obtain sequences located within 1 kb upstream of each transcription strating site (TSS) (http://rsat. scmbb.ulb.ac.be/rsat/). Sequences were masked for the presence of repeats and coding sequences, as well as redundant sequences due to alternative transcripts were also avoided. Sequences obtained were finally scanned for the presence of specific functional DNA motifs by using the Clover algorithm and the library of motifs provided by TRANSFACH Profes- sional 2009.2. For each sequence in both datasets, a P value was estimated as probability of obtaining a raw score of same size or greater merely by chance, using a shuffled version of the target sequence as control and a very stringent pvalue cut-off of 0.001. Western Blotting Analysis ES cells and EB lysates were prepared in RIPA lysis buffer, separated by 10% SDS/PAGE and transferred onto PVDF membranes. Western blots were performed using antibodies against b-Galactosidase (dilution 1:2000; Chemicon), p21 (dilution 1:200; Abcam), p27 (M-197) (dilution 1:200; Santa Cruz), p57 (H- 91) (dilution 1:200; Santa Cruz) and actin (dilution 1:10000; Sigma-Aldrich). Detection of proteins was accomplished using Horseradish-peroxidase-conjugated secondary antibodies and enhanced chemioluminescence (ECL) purchased from Amersham. Scanning densitometry was performed using the ImageQuant software (Molecular Dynamics). PLoS ONE | www.plosone.org 11 February 2010 | Volume 5 | Issue 2 | e9029