## Figure S1A. Blood glucose levels in mice after glucose injection Blood glucose (mm/l) 25 2 15 1 5 # 15 3 6 3+3 Time after glucose injection (min) #
Figure S1B. α-kg levels in mouse livers after glucose injection Abundance -KG 4 3 2 1 17.2 17.3 17.4 17.5 Elution time (min) Abundance Heptadecanoic acid 4 3 2 1 21.3 21.6 Elution time (min) Peak Area (Normalized by heptadecanoic acid).7.6 -KG.5.4.3.2 y =.3382x.124.1 R² =.9998 1 2 Actual quantity (Normalized by heptadecanoic acid) Mouse liver after glucose injection Elution time (min)
# ##Figure S1C. Global 5hmC levels in mouse tissues after glucose injection Mouse liver after glucose injection Genomic DNA (ng) Relative 5hmC 3 2 1 Liver # ## 15 3 6 3+3 Time after glucose injection (min) Mouse kidney after glucose injection Genomic DNA (ng) Relative 5hmC 4 3 2 1 Kidney 15 3 6 3+3 Time after glucose injection (min) Mouse muscle after glucose injection Genomic DNA (ng) 21 22 23 24 5 Fasted, min 6 7 A 9 1 11 12 13 15 16 15 min 3 min 8 19 2 1 2 3 4 6 min 3+3 min 21 22 23 24 5 6 7 9 1 11 12 13 15 16 8 19 2 1 2 3 4 Relative 5hmC 2.5 2. 1..5. Muscle P=.6.s. #n#p=.7 15 3 6 3+3 Time after glucose injection (min)
Figure S1D. Quantification of various cytosine derivatives by LC-MS/MS analysis Actual quantity (normalized by ddc) Actual quantity (normalized by ddc) Actual quantity (normalized by ddc) Actual quantity (normalized by ddc)
Figure S1E. Global 5mC and 5fC levels in mouse livers after glucose injection 2.5 (By LC-MS/MS) Relative 5mC (liver) 2. 1..5. 15 3 6 3+3 Time after glucose injection (min) Relative 5fC (liver) 2.5 2. 1..5 P=.5. 15 3 6 3+3 Time after glucose injection (min)
Figure S1F. The expression and subcellular localization of Tet2 or Tet3 in mouse livers after glucose injection Relative mrna expression 2. 1..5. Tet2 Tet3 3 6 3+3 Time post glucose injection (min) Time post glucose injection Tet3 Tet2 β-actin Cytosolic Fraction Nuclear Fraction Time post glucose injection min 3 min 6 min 3+3 min min 3 min 6 min 3+3 min Tet3 WB Lamin A/C -Tubulin
Figure S1G. Global 5hmC and 5mC levels in mouse livers after glutamine or glutamate injection Mouse liver after glutamine or glutamate injection -5hmC Methylene Blue Genomic DNA (ng) 3 3 Relative 5hmC (liver) (By dot-blot) 2 1 15 3 6 Relative 5hmC (liver) (By dot-blot) 2 1 15 3 6 Time after glutamine injection (min) Time after glutamate injection (min) 2. 2. Relative 5mC (liver) (By LC-MS/MS) 1..5 P=.6 Relative 5mC (liver) (By LC-MS/MS).. 15 3 6 15 3 6 Time after glutamine injection (min) Time after glutamate injection (min) 1..5
Figure S1H. 5hmC and 5mC levels in the selected region of Gck gene in mouse livers after glucose injection 4 5hmC T=min 5hmC in Gck by hmedip-seq 1kb 4 T=3min 4 T=6min 4 5mC T=min 5mC in Gck by MeDIP-seq 5kb 4 5mC T=min 1kb 4 T=3min 4 T=3min 4 T=6min 4 T=6min Relative 5mC enrichment (MeDIP-qPCR) 1..5. p=.6 3 6 3+3
Figure S1I. 5hmC and 5mC levels in a control region of Gck gene in mouse livers after glucose injection 5hmC 3 T= min 3 T=3 min 3 T=6 min Relative enrichment of 5hmC 1..5. 3 6 3+3 Time post glucose injection time (min) Relative enrichment of 5mC 1..5. 3 6 3+3 Time post glucose injection time (min)
Figure S1J. The mrna expression of glycolytic or gluconeogenic genes in mouse livers after glucose injection Relative mrna expression Relative mrna expression Relative mrna expression 2. 1..5. 3 2 1 1..5. G6pase Lpk Pck1 G6Pase LPK PEPCK 3 6 3+3 Time post glucose injection (min)
SUPPLEMENTARY FIGURE LEGENDS Figure S1A. Blood glucose levels in mice after glucose injection. Over-night fasted mice received single or repeated intraperitoneal injection(s) of glucose (1g/kg body weight). Blood glucose was measured at the indicated time points after glucose injection(s). Data are represented as the mean ± S.D. (n=2-5 per group). p <.5, p <.1, p <.1 for comparison with t= min; ## p <.1, ### p <.1 for the indicated comparison; =not significant. Figure S1B. α-kg levels in mouse livers after glucose injection Metabolites were extracted from mouse tissues as described in the Method, and detected for α-kg levels by GC-MS analysis (upper panel). The relative levels of α-kg were calculated using an internal standard heptadecanoic acid (C17:). Data shown are representative of the abundance of α-kg and heptadecanoic acid in mouse livers at the indicated time points after glucose injection (lower panel). Figure S1C. Global 5hmC levels in mouse tissues after glucose injection Genomic DNA was extracted from mouse tissues at the indicated time points after glucose injection. Global 5hmC levels in the liver, kidney, and muscle were determined by dot-blot analysis. In addition, genomic DNA was also spotted and stained with.2% methylene blue in.3 M sodium acetate (ph=5.2) to control equal loading. Data are represented as the mean ± S.D. (n=2-5 per group). p <.5, p <.1 for comparison with t= min; # p <.5, ## p <
.1 for the indicated comparison; =not significant. Figure S1D. Quantification of various cytosine derivatives by LC-MS/MS analysis Dideoxycytidine (ddc), 5mC, 5hmC, 5fC, and 5caC were subjected to LC-MS/MS in the selected reaction monitoring (SRM) scan mode. The typical LC-MS/MS chromatograms of each standard (1 pg) are shown, with ddc (1 pg) being added as an internal control. Standard curves for 5mC, 5hmC, 5fC, and 5caC (1-1 pg) were generated and a good linearity was achieved for these cytosine derivatives. Figure S1E. Global 5mC and 5fC levels in mouse livers after glucose injection Genomic DNA was extracted from mouse livers at the indicated time points after glucose injection, and determined for global levels of 5mC and 5fC by LC-MS/MS. Data are represented as the mean ± S.D. (n=3 per group). p <.5 for comparison with t= min; =not significant. Figure S1F. The expression and subcellular localization of Tet2 or Tet3 in mouse livers after glucose injection The mrna and protein expressions of Tet2 and Tet3 in mouse livers at the indicated time points after glucose injection were determined by qrt-pcr analysis and western-blot, respectively. Moreover, the subcellular localization of Tet3 was determined in isolated cytosolic and nuclear fractions from mouse livers at the indicated time points after glucose
injection. Figure S1G. Global 5hmC and 5mC levels in mouse livers after glutamine or glutamate injection Over-night fasted mice received single intraperitoneal injection of L-glutamine (1.2 mg/g body weight) or glutamate (1.2 mg/g body weight). Genomic DNA was extracted from mouse livers at the indicated time points after glutamine or glutamate injection, and global levels of 5mC and 5hmC were determined by dot-blot and LC-MS/MS analysis. Data are represented as the mean ± S.D. (n=3 per group). p <.5, p <.1 for comparison with t= min; =not significant. Figure S1H. 5hmC and 5mC levels in the selected region of Gck gene in mouse livers after glucose injection An enlarged drawing for hmedip-seq peaks in the Gck gene visualized in IGV, as labelled by a red-dotted box in Figure 1E. One region (chr11:5948151bp-5948348bp) which was chosen for hmedip-qpcr verification in Figure 1F was labeled by a red line (upper panel). Moreover, MeDIP-seq peaks in the Gck gene were visualized in IGV (lower panel). One region (chr11:5948151bp-5948348bp) which was chosen for MeDIP-qPCR verification at the bottom was labeled by a red line. Data are represented as the mean ± S.D. (n=3 per group). p <.5 for comparison with t= min.
Figure S1I. 5hmC and 5mC levels in a control region of Gck gene in mouse livers after glucose injection A control region with similar GC content as the selected region in Figure 1 was labeled by a red line, and 5hmC and 5mC levels in the control region were determined by hmedip-qpcr and MeDIP-qPCR, respectively. Data are represented as the mean ± S.D. (n=3 per group). =not significant. Figure S1J. The mrna expression of glycolytic or gluconeogenic genes in mouse livers after glucose injection The mrna expression of G6pase, Lpk, and Pck1 in mouse livers at the indicated time points after glucose injection was determined by qrt-pcr analysis. Data are represented as the mean ± S.D. (n=3 per group). p <.5; p <.1 for comparison with t= min; =not significant.