hours incubation at 37uC in a humidified chamber, absorbance was recorded at 570 nm. Triplicate analysis was applied to each time point. Population doubling time assay: 105 cells was plated on 1 well of 6-well plate and counted by SSEA-1 positive population after 24 and 48 hours later. Briefly, ES cells were harvested by trypsin and washed with PBS prior to SSEA-1 (mouse monoclonal 1:200 dilution) antibody incubation for 2 hours. After washing with PBS twice, cells were incubated with FITC-conjugated secondary antibody for 1 hour. Finally, cells were washed twice and resuspended in PBS before FACS analysis. The population (%) of positive SSEA-1 stained cells was compared to unstained cells, FITC-isotype control, undifferentiated ES cells, and cells stained with secondary antibody alone. The SSEA-1 positive cell numbers were subjected to the calculation of ’’cell population doubling time‘‘ with the equation Yend = Ystart62 (t/T). Supporting Information Figure S1 Identification of Myc binding sites in ES cells. In addition to the log2 ratio .2 we also applied the widely used ACME/R anaylsis which employs a sliding window = 500 b.p. and a threshold = 95% across gene promoters to determine significance (p-value) of each probe. Our identification of Myc binding sites is based on significant peaks (ACME: p-value ,0.0001) that take into account binding events on neighboring probes. Shown are a few typical promoters in which are plotted log2 (NimbleGen enrichment ratio scale on left ; probes = green dots) and -log10 (ACME p value in red. Scale is shown on right of each figure). Found at: doi:10.1371/journal.pone.0007839.s001 (0.15 MB PDF) Figure S2 Validation of anti-Myc ChIP-chip results in the R1 mES cell line We randomly selected 44 genes for validation and confirmed the enrichment of 44 genes on the array by qChIP- PCR. Known Myc target genes such as Bmi1 served as a positive control. Genes Evx2 and Sox21 not identified in our ChIP-chip analysis and not previously reported as c-Myc targets were used as negative controls for validation by qChIP-PCR. Bar heights represent the mean ‘‘Fold’’ enrichment from 2 independent sets of anti-Myc ChIP-enriched versus total input genomic DNA. Six genes were not consistent in both our ChIP-chip assay and qChIP- PCR. From this we calculate the average ‘‘false discovery rate’’ in R1 ES cells to be 11.3%. Found at: doi:10.1371/journal.pone.0007839.s002 (0.01 MB PDF) Figure S3 Determination of ES cells proliferation rate in response to c-Myc level. c-Myc levels were manipulated by Lentiviral-delivered overexpression or shRNA knock-down of c- Myc in ES cells. (A) BrdU labeling of a representative ES colonies. (B) Mean growth rate was measured by MTT assay. Mean was measured from triplicate experiments. (C) Mean population doubling time obtained from 4 independent experiments. Found at: doi:10.1371/journal.pone.0007839.s003 (0.03 MB PDF) Figure S4 Sox2 and HirA protein expression in mES cells. Antibodies against Sox2 and HirA were used for immunoblots prepared from mES cells infected with empty lentiviral vector alone (WT; left panel); with a lentiviral vector expression shRNA against c-myc (middle panel); or with a lentiviral vector expressing c-myc (right panel). c-tubulin was used for loading control. Found at: doi:10.1371/journal.pone.0007839.s004 (0.04 MB PDF) Myc Function in ES Cells Figure S5 H3-K27me3 (red) and H3-K4me3 (green) patterns at promoters of genes activated following c-Myc overexpression. DNA immunprecipitated with anti-H3-K4me3 or anti-H3- K27me3 was applied to the custom-designed array (see text). Enrichment ratios (log2 scale) for ChIP-enriched versus total input genomic DNA for 427 genes were processed by ACME and assigned p-values (-log10; y axis) identifying significant sites were plotted (see Methods). Red peaks present H3-K27me3 and green peaks present H3-K4me3. Found at: doi:10.1371/journal.pone.0007839.s005 (1.49 MB PDF) Figure S6 H3-K27me3 (red) and H3-K4me3 (green) patterns at promoters of genes repressed following c-Myc overexpression. DNA immunprecipitated with anti-H3-K4me3 or anti-H3- K27me3 was applied to the custom-designed array (see text). Enrichment ratios (log2 scale) for ChIP-enriched versus total input genomic DNA for 427 genes were processed by ACME and assigned p-values (-log10; y axis) identifying significant sites were plotted (see Methods). Red peaks present H3-K27me3 and green peaks present H3-K4me3. Found at: doi:10.1371/journal.pone.0007839.s006 (1.14 MB PDF) Figure S7 H3-K27me3 (red) and H3-K4me3 (green) patterns at promoters in which bivalency is partly lost upon c-Myc overexpression. DNA immunprecipitated with anti-H3-K4me3 or anti-H3-K27me3 was applied to the custom-designed array (see text). Enrichment ratios (log2 scale) for ChIP-enriched versus total input genomic DNA for 427 genes were processed by ACME and assigned p-values (-log10; y axis) identifying significant sites were plotted (see Methods). Red peaks present H3-K27me3 and green peaks present H3-K4me3. Found at: doi:10.1371/journal.pone.0007839.s007 (4.21 MB PDF) Figure S8 H3-K27me3 (red) and H3-K4me3 (green) patterns at promoters in which bivalency is gained upon c-Myc overexpres- sion. DNA immunprecipitated with anti-H3-K4me3 or anti-H3- K27me3 was applied to the custom-designed array (see text). Enrichment ratios (log2 scale) for ChIP-enriched versus total input genomic DNA for 427 genes were processed by ACME and assigned p-values (2log10; y axis) identifying significant sites were plotted (see Methods). Red peaks present H3-K27me3 and green peaks present H3-K4me3. Found at: doi:10.1371/journal.pone.0007839.s008 (1.11 MB PDF) Figure S9 H3-K27me3 (red) and H3-K4me3 (green) patterns at promoters showing only small or no changes in H3-K27me3 and H3-K4me 3 pattern. DNA immunprecipitated with anti-H3- K4me3 or anti-H3-K27me3 was applied to the custom-designed array (see text). Enrichment ratios (log2 scale) for ChIP-enriched versus total input genomic DNA for 427 genes were processed by ACME and assigned p-values (2log10; y axis) identifying significant sites were plotted (see Methods). Red peaks present H3-K27me3 and green peaks present H3-K4me3. Found at: doi:10.1371/journal.pone.0007839.s009 (5.05 MB PDF) Figure S10 c-Myc binding sites on a subset of gene promoters in mES cells. c-Myc levels were manipulated by Lentiviral-delivered overexpression or knock-down of c-Myc in mES cells followed by anti-Myc ChIP-chip analysis on custom- designed arrays (see text). Unprocessed enrichment ratios (log2 scale) present ChIP-enriched versus total input genomic DNA (crosslinked, sonicated and processed identically to the ChIP sample) (y axis) for all probes PLoS ONE | www.plosone.org 11 November 2009 | Volume 4 | Issue 11 | e7839