hesc Otx2 B-T Sox17 Actin M r (K)

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1 DOI:.38/ncb357 In the format provided by the authors and unedited. a Nanog OCT b hesc hipsc hipsc SSEA TRA -6 c hesc hipsc Nanog OCT Otx hesc SSEA TRA -6 B-T d f Sox7 M r (K) M r (K) Actin Actin e OCR (pmol/min/μg protein) 3 hdf hesc hipsc Oligo FCCP Anti/Rot g M r (K) IP: acetyl-k hdf hesc 6 8 Time (min) 36 - Supplementary Figure Warburg-like effect in hescs and hipscs compared to hdfs. (a) Human ESCs (H9) and hipscs cultured under feeder-free condition were stained with specific antibodies against pluripotency markers (e.g., Oct, Nanog, SSEA, and TRA-6) along with Hoechst nuclear staining. Scale bar, μm. (b) Representative pictures of hescs and hipscs. Scale bar, μm. (c) In vitro spontaneous differentiation of hescs and hipscs by culturing in serum-free ITSFn medium for 7 days. Immunostaining images (first and second row panels) show lineage specific markers for ectoderm (Otx), mesoderm (Brachyury; B-T), and endoderm (Sox7). Scale bar, μm. (d) Intracellular ATP levels were significantly lower in hipscs and hescs than in the original fibroblasts. (mean ± s.e.m., n=3 biologically independent experiments, p<.5, two-tailed unpaired Student s t-test). (e) Mitochondrial bioenergetics of parental hdfs and hipscs as well as hescs assessed by Seahorse XF analyzer. (mean ± s.d., n= biologically independent experiments). (f) Expression levels of glucose transporters (GLUTs) including GLUT to GLUT7 in hdfs and hipscs as well as hescs. (mean ± s.e.m., n=3 biologically independent experiments, p<.5; p<.; p<.5; p<., one-way ANOVA with Bonferroni post-test). (g) Immunoprecipitation of hdf and hescs proteins using antibodies against acetyl-lys, followed by LC-MS/MS analyses to identify acetylated proteins. Statistics source data are in Supplementary Table 9. Unprocessed original scans of blots (d,g) are shown in Supplementary Fig Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 Peptide spectrum for tubulin Peptide spectrum for Enolase Peptide spectrum of Fructose-bisphosphate aldolase Peptide spectrum of Pyruvate kinase isozymes M/M Peptide spectrum of Glyceraldehyde-3- phosphate dehydrogenase Peptide spectrum of ATP synthase Peptide spectrum for phosphoglycerate kinase Supplementary Figure CID spectra for the acetylated proteins shown in Supplementary Fig. g and Supplementary Table. Peptides for tubulin, Fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, enolase, pyruvate kinase isozymes M/M and ATP synthase were detected via combination of IP and LC-MS/MS analyses. IP was performed with anti-acetyl-lys antibody. 7 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

3 a Expression Value (Log) SIRT b c d Relative expression Somatic cells hescs Relative expression Somatic cells hescs Relative expression Somatic cells hescs e Relative expression Somatic cells hescs f Relative expression Somatic cells hescs Supplementary Figure 3 Meta-analysis of Sirtuin family expression in hescs. (a) Representative data showing SIRT expression changes between different cells. SIRT downregulation was observed in hpscs compared to differentiated cells and original fibroblasts. (b-f) Mean value scatter plot of expression levels of SIRT3 (b), SIRT (c), SIRT5, (d) SIRT6 (e), and SIRT7 (f) in hesc lines (n=5) and normal somatic cell lines (n=5) using results from a Database search ( All cell lines information is shown in Supplementary Table 5. (mean ± s.e.m., two-tailed unpaired Student s t-test) Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

4 a [SIRT-TA-EGFP] b Relative mrna levels 6 hesc hdf SIRT w/o Dox SIRT with Dox c w/o TGFβ With TGFβ d Dox SIRT-GFP -GFP Relative mrna levels SIRT AldoA ENO PGK 5 SIRT AldoB ENO PGK GAPDH AldoC ENO3 Supplementary Figure Characterization of inducible SIRT-GFP H9 hescs. (a) Plasmid map of doxycycline (Dox) inducible SIRT-EGFP overexpression vector. (b) Expression levels of pluripotency markers in wild-type hescs, hdfs, and SIRT-GFP hescs with or without Dox. (mean ± s.d., n=3 biologically independent experiments, p<.5, one-way ANOVA with Bonferroni post-test). (c) Effects of TGF-β on spontaneous differentiation of SIRT-GFP hescs. Cells were maintained in hesc culture conditions with or without TGF-β. TGF-β was added for days and cells were immunostained for expression of the endodermal marker Sox7; Scale bar, μm. (d) Expression levels of glycolytic enzymes in SIRT-GFP hescs with or without Dox analyzed by qrt-pcr. (mean ± s.d., n=3 biologically independent experiments, p<.5, two-tailed unpaired Student s t-test). Statistics source data are in Supplementary Table Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

5 a [AldoA] b [AldoA+SIRTKD] e Relative AldoA activity (Fold) 3 WT KQ K3Q c [AldoA] d [AldoA+SIRTKD] f 3 SIRTKD Relative AldoA activity (Fold) AldoA-Myc WT KR K3R Supplementary Figure 5 Effects of altered SIRT expression on acetylation of AldoA. (a-d) Detection of AldoA K (a,b) and K3 (c,d) acetylation by mass spectrometry analysis. indicates the acetylation site. (e) Myc-tagged AldoA, AldoA KQ, and AldoA K3Q were each expressed in 93T cells. AldoA proteins were purified by IP with Myc antibody, and specific activity for AldoA was determined. (mean ± s.d., n=3 biologically independent experiments, p<.5, one-way ANOVA with Bonferroni post-test). (f) Myc-tagged AldoA, AldoA KR, and AldoA K3R were each expressed in 93T cells co-expressing SIRT shrna (SIRTKD). AldoA proteins were purified by IP with Myc antibody and specific activity for AldoA was determined. (mean ± s.d., n=3 biologically independent experiments, p<.5, one-way ANOVA with Bonferroni post-test). Statistics source data are in Supplementary Table Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

6 a ECAR (mph/min/μg protein) b ECAR (mph/min/μg protein) g h OCR (pmol/min/μg protein) Number of cells (x ) 3 Glucose Oligo -DG Glucose Oligo -DG Time (min) w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox Basal Glycolytic Rate H7 w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox Glycolytic Capacity H9 Glycolytic Reserve 3 6 Days Number of cells (x ) 8 6 c d ECAR (mph/min/μg protein) ECAR (mph/min/μg protein) OCR (pmol/min/μg protein) Time (min) 8 Basal Glycolytic Rate hipsc- 6 Days w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox Glycolytic Capacity H7 Number of cells (x ) 8 6 Glycolytic Reserve 3 e f ECAR (mph/min/μg protein) ECAR (mph/min/μg protein) OCR (pmol/min/μg protein) hipsc- 3 6 Days Time (min) Basal Glycolytic Rate w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox Glucose Oligo w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox Glycolytic Capacity -DG hipsc- Glycolytic Reserve 3 w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox Supplementary Figure 6 Metabolic and functional characterization of hpsc lines following SIRT overexpression. (a,c,e) Glycolytic bioenergetics of wild type () and inducible SIRT-GFP cell lines from H7 hescs (a) and two ipsc lines (c,e) with or without Dox were assessed by XF analyzer. (mean ± s.d., n=3 biologically independent experiments). (b,d,f) Basal glycolytic rate, glycolytic capacity and glycolytic reserve of mock and SIRTOE from H7 hescs (b) and two ipsc lines (d,f) with or without Dox are shown in (a,c,e), respectively. (mean ± s.d., n=3 biologically independent experiments, p<.5; p<., one-way ANOVA with Bonferroni post-test). (g) OCRs for two hesc lines (H9 and H7) and the hipsc- line with or without Dox are shown. : w/o Dox, : with Dox, 3: SIRTOE w/o Dox, : SIRTOE with Dox. (mean ± s.d., n=3 biologically independent experiments, p<.5; p<.5, one-way ANOVA with Bonferroni post-test). (h) Cell proliferation of mock and SIRTOE from H7 hescs and two independent ipsc lines (hipsc- and hipsc-) with or without Dox were analyzed by determining cell numbers every days under ESC culture conditions. (mean ± s.d., n=3 biologically independent experiments, p<.; p<.5, two-way ANOVA with Bonferroni post-test). Statistics source data are in Supplementary Table Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

7 a b c e Relative mrna levels (Fold) Relative mrna levels (Fold) Lactate (nmol/μg protein/day) Oct 3 Pax6 3 3 d ECAR (mph/min/μg protein) D D D D3 D Nanog 3 B-T 3 3 D D D D3 D Rex 3 Sox7 D D D D3 D D D D D3 D 3 f < >5 hipsc- hipsc- Pax6 Map GFAP AADC Foxa Sox7 AFP CK8 CK8 Msx B-T Pax6 Map GFAP AADC Foxa Sox7 AFP CK8 CK8 Msx B-T D D3 D6 D9 D Supplementary Figure 7 SIRT influences metabolic signatures of early differentiation potential of hipscs. (a-c) Inducible SIRTOE hipsc- cells with or without Dox were induced to differentiate spontaneously by culturing in serum-free ITSFn medium for up to days, and gene expression levels of pluripotency markers (Oct, Nanog, and Rex) (a), early-differentiation markers (Pax6, Brachyury (B-T), and Sox7) (b) and SIRT (c) were determined by qrt-pcr. (mean ± s.d., n=3 biologically independent experiments, p<.5, one-way ANOVA with Bonferroni posttest). (d) Glycolytic bioenergetics of mock and SIRTOE hipsc- cells with or without Dox were assessed using the Seahorse XF analyzer. (mean ± s.d., n=3 biologically independent experiments, p<.5, one-way ANOVA with Bonferroni post-test). (e) Extracellular lactate production of mock and SIRTOE hipsc- cells with or without Dox. (mean ± s.d., n=3 biologically independent experiments, p<.5; p<., one-way ANOVA with Bonferroni post-test). (f) Heatmaps depicting gene expression levels of markers representing ectoderm (Pax6, Map, GFAP and AADC), endoderm (Foxa, Sox7, AFP, CK8 and CK8), and mesoderm (Msx and B-T) in wild type () and inducible SIRTOE hipsc lines including hipsc- and hipsc- with or without Dox for up to days under differentiation conditions. (n= biologically independent experiments). : w/o Dox, : with Dox, 3: SIRTOE w/o Dox, : SIRTOE with Dox. Statistics source data are in Supplementary Table Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

8 a c 6 d 6 OCR/ECAR OCR changes after FCCP injection b e OCR (pmol/min/μg protein) 5 M r (K) - 5 w/o Dox with Dox SIRTOE w/o Dox SIRTOE with Dox 6 8 Time (min) Oligo FCCP Anti/Rot +SIRT (shrna) SIRT Actin SIRTKD f M r (K) - +SIRT SIRT SIRT Actin g OCR (pmol/min/μg protein) h OCR (pmol/min/μg protein) Oligo FCCP Anti/Rot 6 8 Time (min) Oligo FCCP Anti/Rot 6 8 Time (min) w/o Dox (D3) with Dox (D3) Y only w/o Dox (D3) Y only with Dox (D3) Y+SIRTOE w/o Dox (D3) Y+SIRTOE with Dox (D3) w/o Dox (D6) with Dox (D6) Y only w/o Dox (D6) Y only with Dox (D6) Y+SIRTOE w/o Dox (D6) Y+SIRTOE with Dox (D6) Supplementary Figure 8 Effects of altered SIRT expression on metabolic reprogramming and ipsc generation. (a) Plasmid map of doxycycline (Dox) inducible SIRT-EGFP overexpression vector. (b) OCR for hdfs infected with wild type () or inducible SIRT-GFP (SIRTOE) with or without Dox at day 3 post transfection. (mean ± s.d., n=3 biologically independent experiments). (c,d) OCR/ECAR ratio (c), and relative OCR changes after FCCP injection (d) from and SIRTOE with or without Dox are shown in (b). (mean ±s.d., n=3 biologically independent experiments). (e,f) Effects of SIRTKD or OE on ipsc generation. Upper: Efficiency of SIRTKD or OE was confirmed by western blotting with anti-sirt antibody. Lower: Representative pictures of AP-positive colonies at day post-transduction. (mean ± s.e.m., n=3 biologically independent experiments, p<.5, two-tailed unpaired Student s t-test). (g,h) OCR in hdf infected with Y and/or SIRTOE at 3 (g) or 6 (h) days after transfection. (mean ± s.d., n=3 biologically independent experiments). Statistics source data are in Supplementary Table 9. Unprocessed original scans of blots (e,f) are shown in Supplementary Fig Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

9 Fig. a hdf ipsc hesc Acetyl-α-tubulin hdf ipsc hesc Acetyl-α-tubulin hdf ipsc hesc Acetyl-α-tubulin α-tubulin α-tubulin α-tubulin Fig. d hdf ipsc hesc SIRT hdf ipsc hesc SIRT hdf ipsc hesc SIRT SIRT SIRT SIRT Supplementary Figure 9 Unprocessed original scans of Western blots. For some experiments (Figs. a,d,i, b,c,e,f, 3a, 8b,e, and Supplementary Fig. d,g), the independent repeats marked No. and 3 are included. In case of Aldolase A (see Input from Fig. b,c,e,f), additional non-specific bands were observed probably due to the use of different antibody lot numbers Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

10 Fig. i Days Days Days SIRT - SIRT - SIRT Days 8 Days 8 Days 8 SIRT SIRT SIRT Days 8 Days 8 Days 8 Fig. a SIRT 7 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

11 Fig. b SIRTOE SIRTOE SIRTOE [AcK IB from Aldolase A IP] 5 - SIRTOE SIRTOE SIRTOE [AcK IB from PGK IP] SIRTOE SIRTOE SIRTOE [AcK IB from Enolase IP] SIRTOE SIRTOE SIRTOE [AcK IB from GAPDH IP] Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

12 Fig. b SIRTOE SIRTOE SIRTOE Aldolase A (Input) PGK (Input) Enolase (Input) GAPDH (Input) (Input) 7 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

13 Fig. c SIRTOE SIRTOE SIRTOE [Aldolase A IB from AcK IP] 5 - SIRTOE SIRTOE SIRTOE [PGK IB from AcK IP] SIRTOE SIRTOE SIRTOE [Enolase IB from AcK IP] SIRTOE SIRTOE SIRTOE [GAPDH IB from AcK IP] Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

14 Fig. c SIRTOE SIRTOE SIRTOE Aldolase A (Input) PGK (Input) Enolase (Input) GAPDH (Input) (Input) 7 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

15 Fig. e SIRTOE SIRTOE SIRTOE [AcK IB from Aldolase A IP] 5 - SIRTOE SIRTOE SIRTOE [AcK IB from Enolase IP] SIRTOE SIRTOE SIRTOE [SIRT IB from Aldolase A IP] SIRTOE SIRTOE SIRTOE [SIRT IB from Enolase IP] Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

16 Fig. e SIRTOE SIRTOE SIRTOE Aldolase A (Input) SIRTOE SIRTOE SIRTOE Enolase (Input) 5 - SIRTOE SIRTOE SIRTOE (Input) Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

17 Fig. f [AcK IB from Aldolase A IP] hdf SIRTKD hdf SIRTKD hdf SIRTKD [AcK IB from PGK IP] - hdf SIRTKD hdf SIRTKD hdf SIRTKD [AcK IB from Enolase IP] - hdf SIRTKD hdf SIRTKD hdf SIRTKD [AcK IB from GAPDH IP] hdf SIRTKD hdf SIRTKD hdf SIRTKD Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

18 Fig. f Aldolase A (Input) - hdf SIRTKD hdf SIRTKD hdf SIRTKD PGK (Input) - Enolase (Input) - GAPDH (Input) SIRT (Input) (Input) Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

19 Fig. 3a SIRTKD AcK Myc SIRTKD AcK Myc SIRTKD AcK Myc Fig. 7e Fig. 7f SIRT SIRT Fig. 8b 5 c 367 SIRT 5 c 367 SIRT 5 c 367 SIRT Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

20 Fig. 8e Scr 5p 3p SIRT Scr 5p 3p SIRT Scr 5p 3p SIRT Supplementary Fig. d hdf ipsc hdf ipsc hdf ipsc hdf hesc hdf hesc hdf hesc 7 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

21 Supplementary Fig. g M hdf hesc 5 - hdf hesc 5 - hdf hesc Supplementary Fig. 8e Supplementary Fig. 8f SIRT SIRT 7 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

22 Supplementary Table Legends Supplementary Table List of hyperacetylated proteins in hescs includes five glycolytic enzymes Supplementary Table List of hypoacetylated proteins in hescs Supplementary Table 3 Meta-analyses of hpscs and their differentiated cells. (a) hescs, hipscs, and their differentiated cells were grouped and metaanalysis for HAT family was performed using GEOR. The meta-analysis did not reveal any change in HAT expression pattern in hesc and hipsc. GEO accession numbers GSE8633, GSE865, GSE3, GSE39, and GSE979 were used for the analysis. Adj.P.Val indicates P-value adjustment for multiple comparisons. (b) Compiled HAT family data used in this study. Expression levels of each HAT family member shown as up, down, and N/A indicate up-regulated, down-regulated, and no significant change respectively in hescs. Numbers in parentheses indicate the number of changed expression among the 5 different studies. Supplementary Table Meta-analyses of HDAC family gene expression. (a) hescs, hipscs, and their differentiated cells were grouped and meta-analyses performed by GEOR for HDAC family gene expression. (b,c) Compiled data used in this study for HDAC family (b) and Sirtuin family (c). Expression levels of each family member shown as up, down, and N/A indicate up-regulated, down-regulated, and no significant change respectively in hescs. Numbers in parentheses indicate the number of changed expression among the 5 different studies. Supplementary Table 5 (a) List of hesc lines and normal somatic cell lines used for web-based data analyses of Fig. b and Supplementary Fig. 3c-g. (b) List of originally published data sets for all cell lines used for web-based data analyses. Supplementary Table 6 Summary of peptide fragments from acetylated lysine residues identified from control and SIRTKD 93T cells. indicated the site of acetylation detected by LTQ-Orbitrap mass spectrometry. 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