Image analysis and quantification To quantify the fluorescence levels of immunofluorescence- labeled proteins in individual cells, we took sequential images of DAPI and the fluorescence labeled protein using a Leica confocal research microscope with a 63x lens and fixed laser settings per protein. Files were saved in TIFF format with 5126512 resolution. The images were analyzed with Image-Pro 5.1 software (Media Cybernetics). In brief, mean fluorescence intensity levels of the immunofluorescence-labeled protein per cell were measured for the nucleic area as was determined by the DAPI staining for on average 250 cells per quantification and three independent stainings. The distribution of the mean intensity per cell is displayed in histogram figures with same x- axis scale. RNA isolation, real-time QPCR and microarray analysis Ring1b2/Lox;CreERT2 and Ring1b+/+;CreERT2 control ES cells (used as a reference for QPCR and microarray) were cultured under the same conditions and RNA was isolated at the indicated time points. Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufactures protocol followed by DNase treatment and aRNA amplification (Invitrogen). Real-time QPCR conditions and primer sequences are as described elsewhere [21]. Changes in RNA levels following 4-OHT treatment of Ring1b2/Lox;CreERT2 relative to Ring1b+/+;CreERT2 ES cells were determined based on the results of three QPCRs. The oligonucleotide arays were printed at the CMF core facility of the NKI/AvL with mouse Operon microarray version 3 (http://microarrays.nki.nl/download/geneid.html). Detailed in- formation about the labeling and hybridization protocols and array analysis can be found on the NKI/AvL microarray website (http://microarrays.nki.nl/download/protocols.html). In brief, amplified RNA was labeled with ULS Cy5 or Cy3 (Kreatech EA-006, Amsterdam) was pooled and co-hybridized to the microarray (each experiment was performed in dye swap fashion and controlled with self-self hybridization experiments to correct for dye-bias). After 16 hours of hybridization the microarrays were washed and scanned using an Agilent DNA Microarray scanner (Agilent Technologies, Cat# G2505B). ImaGene v6.0 software (BioDiscovery Inc) was used to quantify the RNA expression levels using the tiff images produced by the scanner. The background- corrected intensities from the Cy5- and Cy3 channel were used to calculate log2 transformed ratios. These ratios were normalized using a lowest fit per subarray [66]. A weighted average ratio and confidence level (P-value) was calculated per gene by a NKI platform adjusted error model [67], which was fine-tuned by self- self hybridizations. Significantly differentially expressed genes (outliers) between sample and reference were selected based on their P-value (a gene with a P-value ,0.05 was considered an outlier). The microarray details and results are submitted to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/), ref- erence number E-NCMF-14. Clustering of the microarray data was performed using the bioinformatics software program Genesis [68]. GO classification analysis was performed using bioinfor- matics program BiNGO [36]. Pathway analysis was performed using the bioinformatics software program DAVID based on the KEGG pathway database available online (http://david.abcc. ncifcrf.gov). Ring1b Regulates ES Cell Fate Supporting Information Figure S1 Loss of uH2A after deletion of Ring1b in MEFs. Example of immunofluorescence stainings of Ring1b-/Lox;- CreERT2 MEFs 6 days following 4-OHT treatment showing loss of uH2A in MEFs that have lost Ring1b. Found at: doi:10.1371/journal.pone.0002235.s001 (4.21 MB TIF) Figure S2 Graphical representation of GO categories that are significantly enriched in Ring1b-deficient ES cells. Graphical map showing the hierarchical relations between the significantly enriched GO categories (yellow circles) identified by the BiNGO bioinformatics tool based on the outliers per day of 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells. Found at: doi:10.1371/journal.pone.0002235.s002 (0.32 MB PDF) Figure S3 Ring1b bound H3K4me3 marked genes with CpG- rich promoters retain this mark in NPC and MEFs. Bar graph showing the distribution of the chromatin-state of the Ring1b bound genes with high-CpG content promoters (HCP) in neural progenitor cells (NPC) or MEF that were marked with either H3K4me3 or H3K4me3+H3K27me3 in ES cells. Graph represents the chromatin state of all Ring1b bound genes that are represented on the microarray (‘all’), and only the Ring1b bound genes that are deregulated in Ring1b deficient ES cells (‘dereg’). These data show that most H3K4me3 marked genes, retain this mark in NPC and MEFs. Found at: doi:10.1371/journal.pone.0002235.s003 (0.59 MB TIF) Table S1 Expression Profiling data of Ring1b-deficient ES cells. Excel file of expression profiling data of Ring1b-deficient ES cells, coupled to Ring1b binding data according to Boyer and co- workers (Nature, 2006), based on EntrezIDs. Found at: doi:10.1371/journal.pone.0002235.s004 (7.49 MB XLS) Table S2 BiNGO ontology analysis of genes deregulated in ES cells. Excel file of BiNGO analysis of all outliers based on EntrezIDs per day of 4-OHT treatment of Ring1b-/Lox;- CreERT2 ES cells. Found at: doi:10.1371/journal.pone.0002235.s005 (0.06 MB XLS) Table S3 Pathway analysis of genes transcriptionally deregulat- ed following Ring1b deletion. Excel file of DAVID pathway analysis (selecting the KEGG pathway database option) of all outliers, based on EntrezIDs, per day of 4-OHT treatment of Ring1b-/Lox;CreERT2 ES cells. Found at: doi:10.1371/journal.pone.0002235.s006 (0.02 MB XLS) Acknowledgments We are indebted to all members of the van Lohuizen lab for technical assistance and suggestions, and thank Sophia Bruggeman, Anke Spar- mann, Alexandra Pietersen for critical comments and Anders Lund and Inmaculada Herna´ndez-Muno˜ s for helpful discussions. We greatly appreciate Kathrin Plath for technical assistance and critical comments. We thank Ate Loonsta and Anton Berns for the R26CreERT2 targeting construct and sharing unpublished results. We thank Coenraad Kuijl, Lauran Oomen and Lenny Brocks for assistance with confocal microscopy and immunofluorescence analysis, and members of the NKI microarray core facility for assistance with the microarray analysis. Author Contributions Conceived and designed the experiments: Mv EB Pv JV. Performed the experiments: EB SN MH Pv DH JV. Analyzed the data: LW RK EB MH Pv. Wrote the paper: Mv Pv. PLoS ONE | www.plosone.org 12 May 2008 | Volume 3 | Issue 5 | e2235