Additional Supporting Information may be found in the online version of this article.

STEM_493_sm_SuppFig1.tif1460KSupporting Information Figure 1. Identifying a set of ascorbate-specific alterations. Median beta values for ASC- samples (X axis) are plotted against median beta values for ASC+ samples (Y axis) for all 27,578 probes. A two tail rank sum test (Mann Whitney) identified 2,958 differentially methylated probes at an FDR-adjusted p-value of 0.01 (multiple hypothesis adjustment was performed using Benjamini-Hochberg 1995 step-up method). Of these, 2,205 probes (1,847 genes) were perfect discriminators, i.e. where the lowest of the Asc- Beta values was higher than the highest of the Asc+ (or vice-versa). Perfect discrimination corresponds to an FDR-adjusted p-value of 0.002. Of these 2,205 perfect discriminators, 2,194 (all but 11) were in the direction of down-methylated in the ASC+ samples.
STEM_493_sm_SuppFig2.tif1386KSupporting Information Figure 2. (A) Global distribution of Infinium CpG island probes relative to UCSC knownGeneII gene transcription start sites. (B) Comparison of ascorbate/redox hypomethylated genes (1780) with genes exhibiting r-DMRs (3017) [1] and genes marked by bivalent marks (3292) [19].
STEM_493_sm_SuppFig3.tif27128KSupporting Information Figure 3. Western blot detection of global H3K9 methylation and acetylation patterns in hESCs cultured with or without ascorbate. (A) HES2 hESCs cultured with or without ascorbate for 4 passages (28 days) were assayed by western blotting with antibodies to monomethylated (me1), dimethylated (me2), trimethylated (me3), acetylated (acetylation) H3K9 and histone 3. (B) Densitometry quantification of me1, me2, me3 and acetylation of H3K9 relative to total histone 3 protein (H3).
STEM_493_sm_SuppFig4.tif13914KSupporting Information Figure 4. Evaluation of mRNA expression of hypoxia-inducible factors (HIF1A, HIF1B and HIF 2A) and Jumonji family histone demethylases (JMJD1 and JMJD2) in HES3 hESCs cultured with or without ascorbate for 7 days. For each mRNA the values were normalized to β-actin transcript. Values are mean +/- S.D. and error bars are representative of 3 replicates.(RQ : relative quantitative expression)
STEM_493_sm_SuppTable1.xls22509KSupporting Information Table 1. Excel file with the full data set for all probes in HES2 and HES3 lines.
STEM_493_sm_SuppTable2.pdf248KSupporting Information Table 2. DAVID analysis of the 1847 genes that are perfect discriminators between samples cultured in the absence or presence of vitamin C.
STEM_493_sm_SuppTable3.pdf87KSupporting Information Table 3. Ingenuity analysis of the 1847 ascorbate-responsive differentially methylated genes in HES2 and HES3.
STEM_493_sm_SuppTable4.xls195KSupporting Information Table 4. Excel file with the 891 ascorbate-responsive differentially methylated probes shared between HES2 and HES3.
STEM_493_sm_SuppTable5.xls185KSupporting Information Table 5. Excel file with ascorbate-mediated transcriptional changes. (A) Genes up-regulated in KOSR with vitamin C vs medium without vitamin C. (B) Genes down-regulated in KOSR with vitamin C vs medium without vitamin C.
STEM_493_sm_SuppTable6.xls20KSupporting Information Table 6. Excel file with genes undergoing ascorbate-mediated demethylation showing up or down-regulated gene expression.
STEM_493_sm_SuppTable7.xls117KSupporting Information Table 7. Excel file with genes demethylated by vitamin C, genes exhibiting r-DMRs and genes marked by bivalent marks.
STEM_493_sm_SuppTable8.doc34KSupporting Information Table 8. The 14 of 20 genes that were found to be demethylated with increased passage of hESC (1536 CpG sites (from 371 genes) in 14 independently isolated hES cell lines of various passages by Bibikova et al [22] overlap with the 733 genes found to be commonly demethylated by vitamin C in this study.

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