Figure 10. Validating the Notch1 target genes Sox9, Pax6, Id4, Runx1 and Myf5. Notch1 signaling was induced in ESC and differentiating embryoid bodies by addition of OHT (500 nM). (A) Immunoblot of EB5 NERT ESC (NERT ESCs) and EB5 NERT differentiated embryoid bodies (NERT Mes. 5d) with Sox9, Pax6 and Id4 antibodies. One representative example of three independent experiments is shown. (B) Mesodermal differentiation of Runx1-Venus-NERT ESC or Runx1-Venus control ESC and analysis of the percentage of venus+ and Flk1+ cells by FACS. Error bars indicate standard deviation for three independent experiments. (C) Mesodermal differentiation of Myf5-lacZ- NERT or Myf5-lacZ control ESC and detection of LacZ expression. One representative example of three independent experiments on day 5 is shown. Virtually identical results were obtained when the cells were analyzed 4 or 7 days, respectively, after induction of differentiation. Addition of OHT at day 3 of embryoid body culture similarly yielded in an increased number of lacZ positive cells at days 4, 5 and 7 in the presence of OHT after induction of differentiation. doi:10.1371/journal.pone.0011481.g010 Context-Specific Notch Targets contribute to fine-tuning of the expression of Notch target genes also in mammals (Fig. 12B). In summary, we propose that Notch signaling ensures correct, coordinated and timed specification of stem cells by simultaneously activating LSTFs, differentiation- inhibitory proteins of the Hes/Hey and Id family, Notch receptors and miRNAs in an cell-intrinsic and cell-extrinsic context dependent manner. Materials and Methods Cell Culture The culture of undifferentiated ESC was performed as described in Schroeder et al. [45]. To generate mesodermal cells, ESC were cultured in aMEM containing 15% pre-tested FCS (PAN, Germany), 100 mM 2-mercaptoethanol (differentiation medium) for 4 to 5 days [57] for transcriptome analysis. 56104 undifferentiated ESC per well were cultured in 6 well collagen type IV coated plates (Becton Dickinson, USA). Alternatively, for control of differentiation potential, 16104 undifferentiated ESC were cultured in 6 well plates containing confluent OP9 stromal cells [58]. The culture was then analyzed by flow cytometry for Flk-1+ cells. For the culture of cells in conditions favoring ectodermal or mesodermal differentiation, ESC grown on gelatine were washed and cultured for 4 h in the presence or absence of 1 mM OHT in either ES to ectoderm differentiation medium (GMEM, 10% Knockout Serum Replacement, 1% non essential amino acids, 1 mM sodium pyruvate (all Invitrogen), and 100 mM 2-mercaptoethanol) or in ES to mesoderm differentiation medium (Knockout-DMEM, 10% Knockout Serum Replacement (all Invitrogen), 1% pre-tested FCS (Cambrex), 1% non essential amino acids (Invitrogen), 2 mM L-Glutamine, and 100 mM 2- mercaptoethanol). To block protein synthesis, 50 mg/ml cyclo- heximide (CHX) was added for 4 h. Runx1-Venus ESC [50] and Myf5-LacZ ESC [49] were stably transduced with 10 mg of pCAG-NERTDOP vector or pCAG-IP control vector by electroporation and selected as described in Schroeder et al [45]. For in vitro differentiation of Myf5-LacZ- NERT ESC and Runx1-Venus-NERT ESC, ESC were differ- entiated using the hanging drop method as described [59]. Briefly, ESC were cultivated in hanging drops for 2 days until embryoid bodies were formed. Embryoid bodies were the transferred to bacteriological plates and further cultivated in suspension. Between day 5 and day 7 cells were plated on gelatine-coated tissue culture dishes and further incubated until analysis. ESC were electroporated and selected as previously described [14]. Microarrays RNAs were processed as described previously with minor changes [60] for use on Affymetrix GeneChips Mouse Genome 430 2.0 (Affymetrix, Santa Clara, CA, USA) and analyses were done with dChip Software. RNA from the different cell populations was isolated and treated with DNase I (RNeasy, Qiagen) and used for cDNA synthesis (5 mg total RNA, Expression Analysis Technical Manual, Affymetrix, Santa Clara, CA, USA). cRNA was generated (BioArray High-Yield Transcript Labeling kit, ENZO, Farmingdale, NY, USA) and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 Array (15 mg cRNA, 16 h, 45uC) that contain ,45,000 probe sets of over 39,000 murine transcripts. GeneChip arrays were stained, washed and scanned according to the manufacturer’s specifications. Principle compo- nent analysis (PCA) was performed using the R software (http:// www.r-project.org/; [61]). Scanned GeneChip.DAT files were analyzed by dChip Software (Built date Aug 21, 2008; [62] PLoS ONE | www.plosone.org 14 July 2010 | Volume 5 | Issue 7 | e11481