supplementary information inventory - genes &...

1

Supplementary Information Inventory:

Figure S1, related to Figure 1.

Both fumurate and succinate are competitive inhibitors of -KG-dependent histone

demethylases in vitro with succinate being more potent than fumarate


Treatment with cell permeable fumarate and succinate increases intracellular

accumulation of fumarate and succinate, respectively


Knocking down endogenous FH or SDH increases intracellular levels of fumarate and

succinateand-KG can rescue the effect of FH or SDH knock-down on altering

histone methylations, HIF1, and endostatin levels in a dose-dependent manner


Knocking down FH or SDH reduces the TET-catalyzed 5hmC production in cultured

cells


Knocking down Fh or Sdha leads to accumulation of intracellular fumarate and

succinate in mouse livers


Knocking down Fh or Sdha increases histone methylations and HIF1, and reduces

endostatin and 5hmC levels in mouse livers

2


Knocking down Fh or Sdha may inhibit the enzymatic activity of TETs not by

down-regulating the transcription of TET genes in mouse livers

Figure S8, related to Figure 3 and 5.

Methylene blue staining


Ectopic expression of wild-type FH or SDH, but not the tumor-derived mutants,

decreases intracellular levels of fumarate and/or succinate

Supplemental Figure Legends

Supplemental Table S1

siRNAs targeting human FH and SDHA/B, and mouse Fh and Sdha

Supplemental Table S2

Primer sequences for quantitative RT- PCR

Supplemental Experimental Procedures

Reference

3

Xiao et al. Supplemental Figure S1

Me3

Me2Me1

Me3Me3Me3Me2 Me2

Me2Me2 Me2

Me1Me1

HsKDM4A

Me1

Me3 Me3 Me3

Me1 Me1Me1 Me1

-KG (M) 0 0 0.3 0.6 0.9 1.2 1.8 3

0 0 0.4 0.8 1.2 1.6 2.4 6

0 15 15 15 15 15 15 15 Succinate (mM)

Me3Me3 Me3

Me3 Me3 Me3Me2 Me2 Me2

Me2Me2Me1

Me2Me1 Me1

Me1 Me1 Me1

Me3

Me2Me1

Me3

Me2

Me1

0 15 15 15 15 15 15 15 -KG (M) Fumarate (mM)

Me3

Me2Me2

0 0 0.3 0.6 0.9 1.2 1.8 3

0

20

40

60

80

Me3

rem

ain

ing

(%

) 100

0 15 15 15 15 15 15 15

Me3

rem

ain

ing

(%

)

0

20

40

60

80

100

0 15 15 15 15 15 15 15

0 0 0.4 0.8 1.2 1.6 2.4 6

HsKDM4A

H3K36me3 H3K36me3

-KG (M)

Fumarate (mM)

-KG (M)

Succinate (mM)

Me1

rem

ain

ing

(%

)

0

B

0

20

40

60

80

100-KG (M)

-KG (M)

H3K9me1

CeKDM7A

-KG(M)Fumarate (mM)

-KG(M)Succinate(mM)

50 50 50 50 100 100 100 100

0 1 3 10

Me0

1 3 10

50 50 50 50 100 100 100 100

0 1 3 10 0 1 3 10

Me0

Me1

Me1 Me1

Me0

Me1

Me1 Me1

Me0

Me0

Me0

Me1 Me0 Me0

Me0

Me1

Me0

Me1

Me1

Me0

Me0

Me1 Me1 Me1 Me1 Me1

Me0

Me0

Me0 Me0Me1

Fumarate (mM)

Succinate (mM)

0 1 3 10

1 3 100 0 0

0 0 0 0

A

4

450000

350000

250000

150000

50000 26.40 26.60 26.80 27.00 27.20 26.40 26.60 26.80 27.00 27.20 26.40 26.60 26.80 27.00 27.20

450000

350000

250000

150000

50000

450000

350000

250000

150000

50000


A

B

-KG Succinate Fumarate Asparate1

10

100

1000

10000Con

Methyl-succinateMethyl-fumarate

n.s.

n.s. n.s.

n.s.

n.s.

n.s.**

**

Rel

ativ

e A

bu

nd

ance

(x1

04)

Succinate/-KG Fumarate/-KG0.1

1

10

100Con

Methyl-succinateMethyl-fumarate** n.s.

n.s. **

Met

abo

lite/

-KG

rat

ioA

bu

nd

an

ce

Elution Time (min)

succinate

fumarate

succinate

fumarate succinate

fumarate

Methy-fumarate Methy-succinate Con

5


A

B

Con siFH

Octyl-KG - - + +

H3K79me2

H3K4me1

H3K4me3

H3

Actin

Endostatin

HIF1

FH

H3K9me2

Con siSDHA

Octyl-KG - - + +

Con siSDHB

Octyl-KG - - + +

H3K79me2

H3K4me1

H3K4me3

H3

Actin

Endostatin

HIF1

SDHA

H3K9me2

H3K79me2

H3K4me1

H3K4me3

H3

Actin

Endostatin

HIF1

SDHB

H3K9me2

Actin

SDHA

Actin

FH

Actin

SDHB

Con siFH

#1 #2 #3

Con siSDHA

#1 #2 #3

Con siSDHB

#1 #2 #3 A

bu

nd

an

ce

Con siSDHA #1 siSDHA #2 siSDHA #3

siSDHB #1 siSDHB #2 siSDHB #3

Elution Time (min)

Ab

un

da

nce

Standard Con siFH #1 siFH #2

6


A

B

C

Con shFH

FH

Actin

Con shSDHA

SDHA

Actin

Con shSDHB

SDHB

Actin

Fla

g-T

ET

5h

mC

DA

PI

pMKO

TET1-CD TET1-CM

shFH #2 shFH #3pMKO pMKO

TET2-CD TET2-CM

shFH #2 shFH #3pMKO

pMKO

TET1-CD TET1-CM

shSDHA #2 shSDHA #3 pMKO pMKO

TET2-CD TET2-CM

shSDHA #2 shSDHA #3 pMKO

Fla

g-T

ET

5h

mC

DA

PI

7


A

B

-KG Succinate Fumurate Asparate1

10

100

1000

10000Scramble siFh

**

*

siSdha

**

p=0.07

n.s.n.s.

n.s.n.s.

Rel

ativ

e A

bu

nd

ance

(x1

05)

MS fractionation identification

Elution Time (min)

Ab

un

dan

ce

8


A

B C

HIF1

Endostatin

Sdha

H3K9me2

H3K79me

2

H3K4me1

H3K4me3

Mouse liver Scramble siSdha #2 Scramble siSdha #3

H3

Mouse liver Scramble siFh #2 Scramble siFh #3

HIF1

Endostatin

Fh

H3K9me2

H3K79me

2

H3K4me1

H3K4me3 H3

H3K27me2 H3K27me2

5hmC IHC assay in mouse liver

Scramble siFh #2 Scramble siFh #3

Scramble siSdha #2 Scramble siSdha #3

Genomic DNA (ng) Methylene Blue staining

500 250 100 50

Scramble

siFh #3

Scramble

siFh #2

5hmC dot-blot assay in mouse liver

Scramble

siSdha #3

Scramble

siSdha #2

Genomic DNA (ng) Methylene Blue staining

500 250 100 50

Actin Actin

9


B

A

0

20

40

60

80

100

0

20

40

60

80

100

siFh Scramble siSdhaScramble

Rel

ativ

e F

h m

RN

A le

vel

Rel

ativ

e S

dh

a m

RN

A le

vel

0

0.5

1

1.5

2

2.5 Scramble

siFh

siSdha

Tet1 Tet2 Tet3

Rel

ativ

e T

et m

RN

A le

vel

10


A

B

Genomic DNA (ng) 500 100 50

shFH+TET2-CD

pMKO+TET2-CD

pMKO+TET2-CM

shFH+TET1-CD

pMKO

pMKO+TET1-CD

pMKO+TET1-CM

pMKO


Genomic DNA (ng) 500 250 100 50

shSDHA+TET2-CD

pMKO+TET2-CD

pMKO+TET2-CM

shSDHA+TET1-CD

pMKO

pMKO+TET1-CD

pMKO+TET1-CM

pMKO


Genomic DNA (ng) 500 250 100 50

shSDHB+TET2-CD

pMKO+TET2-CD

shSDHB+TET1-CD

pMKO+TET1-CD

pMKO+TET1-CM

pMKO


Genomic DNA (ng) 500 250 100 50

pMKO

shFH

shFH+WT

shFH+R190H

TE

T1

Genomic DNA (ng) 500 250 100 50

pMKO

shFH

shFH+WT

shFH+R190H

TE

T2

Methylene blue staining Methylene blue staining

Genomic DNA (ng) 500 250 100 50

pMKO

shSDHA

shSDHA+WT

shSDHA+R190H

TE

T1

Genomic DNA (ng) 500 250 100 50

pMKO

shSDHA

shSDHA+WT

shSDHA+R190H

TE

T2


Genomic DNA (ng) 500 250 100 50

pMKO

shSDHB

shSDHB+WT

shSDHB+R190H

TE

T1

Genomic DNA (ng) 500 250 100 50

pMKO

shSDHB

shSDHB+WT

shSDHB+R190H

TE

T2


pMKO+TET2-CM

pMKO

250

11


A

B

C


10

100

1000

pMKO shFH shFH+FHWT shFH+FHR190H

shFH+FHH137R

**

n.s.

*

n.s.

n.s.

**R

elat

ive

Ab

un

dan

ce (

x105

)


10

100

1000

pMKO shSDHA shSDHA+SDHAWT shSDHA+SDHAG555E

shSDHA+SDHAR554W

*n.s.

**n.s.*

n.s.

Rel

ativ

e A

bu

nd

ance

(x1

05)


10

100

1000

pMKO shSDHB shSDHB+SDHBWT shSDHB+SDHBR46Q

shSDHB+SDHBA43P

*

n.s.

*n.s.*

n.s. *

Rel

ativ

e A

bu

nd

ance

(x1

05)

12

Supplemental Figure Legends

Figure S1. Both fumurate and succinate are competitive inhibitors of -KG-dependent

histone demethylases in vitro with succinate being more potent than fumarate

(A) Both fumurate and succinate compete with-KG, directly inhibiting the activity of

-KG-dependent CeKDM7A in vitro, as determined by mass spectrometry (MS).

(B) The half maximal inhibitory concentration (IC50) of fumarate and succinate on

HsKDM4A was determined by MS.

Figure S2. Treatment with cell permeable fumarate or succinate increases

intracellular levels of fumarate and succinate, respectively

(A) Treatment HEK 293T cells with cell permeable methyl-fumarate (2.5mM) or

methyl-succinate (5mM) increases intracellular levels of fumarate and succinate,

respectively, as determined by gas chromatography-mass spectrometry (GC-MS).

(B) Relative levels of fumurate and succinate and the ratios of succinate/-KG and

fumarate/-KG in HEK 293T cells treated with cell permeable fumarate or succinate.

Asparate was used as an internal control. Error bars represent ±SD for triplicate experiments.

**p<0.01 vs. Control (Con); n.s.= not significant.

Figure S3. Knocking down endogenous FH or SDH increases intracellular levels of

fumarate and succinateand-KG can rescue the effect of FH or SDH knock-down on

altering histone methylations, HIF1, and endostatin levels in a dose-dependent

manner

(A) Knocking down FH or SDHA/B in HeLa cells increases intracellular levels of fumarate

and succinate, respectively, as determined by GC-MS. Three different siRNAs were used for

13

each gene. The knock-down efficiency was verified by western-blot.

(B) HEK293T cells with knock-down of FH or SDHA/B were treated with increasing

concentrations of octyl--KG (2.5mM and 5mM). The alterations in histone methylations,

endostatin, and HIF1 levels were determined by western-blot.

Figure S4. Knocking down FH or SDH reduces the TET-catalyzed 5hmC production in

cultured cells

(A) HEK293T cells with stable knock-down of FH or SDHA/B were transiently transfected

with plasmids expressing indicated proteins. The knock-down efficiency was verified by

western-blot.

(B-C) Cells were fixed and stained with specific antibodies against Flag, 5hmC, and DAPI.

Immunofluorescence staining data are shown. Scale bars represent 251m.

Figure S5. Knocking down Fh or Sdha leads to accumulation of intracellular fumarate

and succinate in mouse livers

(A) MS fractionation identification of derivatized fumurate and succinate in mouse livers after

transient knock-down of Fh or Sdha. Mass fragment spectra from the peaks at 14.141 and

14.510 min were identified as the derivatives of succinate and fumarate, respectively (left

panel). The bis-TBDMS succinate-specific 289-m/z fragment and the bis-TBDMS fumarate

287-m/z fragment are shown (middle and right panels).

(B) Relative levels of fumurate and succinate in mouse livers after transient knock-down of

Fh or Sdha were determined by GC-MS. Asparate was used as an internal control. Error

bars represent ±SD for triplicate experiments. *p<0.05 and **p<0.01 vs. Scramble; n.s.= not

significant.

14

Figure S6. Knocking down Fh or Sdha increases histone methylations and HIF1, and

reduces endostatin and 5hmC levels in mouse livers

(A) Transient knock-down of Fh or Sdha increases histone methylations and HIF1and

reduces endostatin levels in mouse livers, as determined by western-blot.

(B) Transient knock-down of Fh or Sdha decreases 5hmC levels in mouse livers, as

determined by immunohistochemistry. Scale bars represent 334m.

(C) Transient knock-down of Fh or Sdha decreases 5hmC levels in mouse livers, as

determined by dot-blot. Genomic DNAs were spotted and stained with methylene blue to

control equal loading.

Figure S7. Knocking down Fh or Sdha may inhibit the enzymatic activity of TETs not

by down-regulating the transcription of TET genes in mouse livers

(A) Verification of the transient knock-down efficiency of Fh or Sdha in mouse livers, as

determined by quantitative real time-PCR.

(B) Knock-down of Fh or Sdha in mouse livers does not down-regulate mRNA expression of

Tet genes, as determined by quantitative real time-PCR. Error bars represent ± SD for

triplicate experiments.

Figure S8. Methylene blue staining

(A-B) HEK293T cells with stable FH or SDHA/B knock-down were transiently transfected

with plasmids expressing indicated proteins. Genomic DNAs were spotted and stained with

methylene blue to control equal loading.

15

Figure S9. Tumor-derived FH or SDH mutants are not functional in fumarate or

succinate metabolism.

(A-C) HEK293T cells with stable FH or SDHA/B knock-down were transiently transfected

with plasmids expressing wild-type FH or SDH as well as tumor-derived FH or SDH mutants.

Relative levels of intracellular fumurate and succinate were determined by GC-MS. Asparate

was used as an internal control. Error bars represent ±SD for triplicate experiments. *p<0.05

and **p<0.01 vs. Scramble; n.s.= not significant.

Table S1. siRNAs targeting human FH and SDHA/B, and mouse Fh and Sdha

Targeting Gene Sense Strand No. Sequence

Human FH siRNA-1 5’-UGCAUUAGCAAUCUUUGGAUCAAGA

siRNA -2 5’- AUAACCACUAAAUUCCUGCCCAAGA

siRNA -3 5’-UUUAAACCUGUACCAACAGCAGUGC

SDHA siRNA-1 5’-GGAGUAAUCGUACUCAUCAAUCCGC

siRNA-2 5’-UAUUCCAGAGUGACCUUCCCAGUGC

siRNA-3 5’-ACCAAAUGCACGCUGAUAAAUCUUC

SDHB siRNA-1 5’-AUAGAUGGCAAAUUUCUUGAUACGG

siRNA-2 5’-UUUCCCUGGAUUCAGACCCUUAGGA

siRNA-3 5’-UUAAACUGAAGCUUUCUUCUCCUUA

Mouse Sdha siRNA 5’- ACCAAAUGCACGCUGAUAAAUCUUC

Fh siRNA 5’- CAAGCTAATGAATGAGTCT

16

TableS2. Primer sequences for quantitative real time-PCR

Gene Strand Sequence

GAPDH Forward 5’- CATGGCCTTCCGTGTTCCTA

Reverse 5’- GCCTGCTTCACCACCTTCTT

β−ACTIN Forward 5’- TCCCTGGAGAAGAGCTACG

Reverse 5’- GTAGTTTCGTGGATGCCACA

Human HOXA2 Forward 5’-ACAGCGAAGGGAAATGTAAAAGC

Reverse 5’-GGGCCCCAGAGACGCTAA

HOXA4 Forward 5’-TCCCCATCTGGACCATAATAGG

Reverse 5’- GCAACCAGCACAGACTCTTAACC

HOXA5 Forward 5’-TCTCGTTGCCCTAATTCATCTTTT

Reverse 5’-CATTCAGGACAAAGAGATGAACAGAA

Mouse Sdha Forward 5’- ATTTCTACTCAATACCCAGTGGTGG

Reverse 5’- CAACAGTATGTGATCGGGTAGGAAA

Fh Forward 5’- CACGCAGGATGCTGTCCCTCTTACT

Reverse 5’- TTCATGGCTCCACTAAGCTCAACCA

Hoxa10 Forward 5’- ATGAGTCAAGGCAGTTCCAAAGGCG

Reverse 5’- TTAAAGTTGGCTGTGAGCTCCCGGA

Hoxa11 Forward 5’- GAGTACGCCATTGAGCCCGCC

Reverse 5’- AGACGCTGAAGAAGAACTCTCGCT

Hoxa13 Forward 5’- CGCCGACAAGTACATGGACACCGC

Reverse 5’- ACCCCCGAGCCCCGGAACTA

17

Supplemental Experimental Procedures

Antibodies

Antibodies to FH, SDHA, SDHB (Abcam, Cambridge, MA), Flag (Sigma-Aldrich,

Shanghai, China), HIF-1 (Novus Biologicals, Littleton, CO), endostatin (Novus Biologicals,

Littleton, CO), histone H3 (Genescript, Piscataway, NJ), H3K4me1, H3K4me3, H3K9me2,

H3K27me2, H3K79me2 (Abcam, Cambridge, MA), H3K36me2 (Active motif, USA), and

actin (GenScript Corp., Piscataway, NJ) were purchased commercially.

Plasmids, cell culture and transient transfection

Wild-type and catalytic mutant TET1/2 and full-length of human FH, SDHA, SDHB were

cloned into Flag- or GFP-tagged destination vectors (pcDNA3.1) according to different

experimental purposes. Point mutations for FH, SDHA and SDHB were generated by

site-directed mutagenesis by using QuickChange Site-Directed Mutagenesis kit

(Stratagene).

All experiments were carried out in HEK293T cells unless specified otherwise. Cells

were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Shanghai,

China) supplemented with 10% newborn bovine serum (Biochrom, Germany). Plasmid

transfection was carried out by the calcium phosphate method for HEK293T. In addition,

HeLa and U87-MG glioblastoma cells were transfected by using lipofectamin 2000

(Invitrogen).

The cell permeable octyl--KG ester, methyl-succinate ester and methyl-fumarate ester

were synthesized, and used to treat cells for 4-6 hours with a final concentration of 5mM,

18

5mM, and 2.5mM, respectively.

GC-MS analysis

Upon harvest, cells were fixed by immediate addition of 1ml 80% (v/v) pre-cold (-80 ºC)

methanol into culture plates. Lyophilized samples were oximated with 20mg/ml

methoxyamine hydrochloride in pyridine at 30 C for 60 min. The samples were then

derivatized at 70 °C for 30 min in 80µl pyridine (Sigma-Aldrich) and 20µl

N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA , Sigma-Aldrich). After

filtration, 3µl of derivatized sample was injected for the GC-MS assay using an Agilent

6890-5973 GC-MS system with a HP-5MS column (30m0.25mm0.25μm). GC oven

temperature was programmed from 60 C to 180 C at 5 C/min and from 180C to 260C at

10C/min. The flow rate of carrier gas was set at 1 ml/min. The mass spectrometer was

operated in the electron impact (EI) mode at 70 eV. Relative concentrations of succinate and

fumuarate were calculated by normalizing succinate (26.48 min) and fumurate (27.06 min)

peak areas to L-threonine (29.58 min), L-serine (29.96 min) and L-phenylalanine (30.74 min)

peak areas. The standard fumarate and succinate were purchased commercially (Sigma,

Shanghai, China).

In addition, mouse livers were rapidly harvested, stored in liquid nitrogen, and then

transferred to -80ºC freezer. Each of frozen tissue samples (100mg) was grinded with liquid

nitrogen into powder, and then homogenized in 1ml 80% (v/v) pre-cold (-80 ºC) methanol.

The homogenate was transferred to a 1.5ml eppdorf tube, held on ice for 30min, and

centrifuged at 12000rpm for 10min at 4 ºC. The supernatant (100 µl) was transferred to a

19

screw-cap glass bottle (CNW 9mm) and dried with vacuum at room temperature (Eppendorf

concentrator 5301). Lyophilized samples were oximated with 20mg/ml methoxyamine

hydrochloride (Sigma-Aldrich) in pyridine at 37ºC for 16-24 hours. The samples were then

derivatized following the same process as above mentioned. Without filtration, 1µl of the

derivatized sample was injected for chromatographic separation on an Agilent 7890-5975

GC-MS system. Injection was performed in the splitless mode at 250°C with a hold time of 3

min followed by continuous venting at 1 ml/min. The Agilent HP-5MS was set at an initial

temperature of 100°C for 3 min and then was increased to 140°C at a rate of 10°C /min, then

further increased to 260°C at a rate of 8°C /min. A heating rate of 10°C/min was then

employed until 310°C was reached. This temperature was then held for 5 min. The mass

spectrometer (70 eV, EI mode) was set up to record the spectrum between 50 and 600 m/z

units.

Retroviral infection and generation of stable cell lines

To generate FH or SDHA/B knocking-down retroviruses, HEK293T cells were plated

40-60% confluent in 90mm-diameter cell culture dishes and then transfected with retroviral

vectors (pMKO) by the calcium phosphate method. At 6 hours post-transfection, the medium

was replaced with fresh DMEM containing 10% newborn bovine serum, and cells were

grown for an additional 18 hours. The conditioned medium containing recombinant

retroviruses was collected, or applied immediately to cells, which had been plated 24 hour

before infection at a density of 10% confluent in 90 mm-diameter cell culture dishes, and

incubated for 24 hours. After double infections, the cells were placed in fresh growth medium

20

and cultured as usual. The selection with 5μg/ml of puromycin was initiated 24 hour after

infection. The newly generated stable cell line was verified by western-blot or quantitative

real-time PCR.

Animals

Male BALB/c mice (6-8 weeks of age, weighing 20-25 g) were purchased from Fudan

Animal Center. Animals were given unrestricted access to a standard diet and tap water.

Animal experiments were performed at Fudan Animal Center in accordance with the animal

ware fare guidelines.

Immunoprecipitation and western-blot

Cells were lysed by 0.5% NP-40 buffer (50mM Tris-HCl, pH 7.5; 150mM NaCl; 0.5%

NP-40) containing protease inhibitor cocktail (Roche, Shanghai, China). Proteins were

affinity purified by incubating cell extract supernatants with Flag beads (Sigma) for 3 hours,

and then were washed with ice-cold PBS (pH 7.4) for three times. Proteins were either

eluted for activity assay by Flag peptide or denatured by SDS loading buffer containing DTT

before western-blot analysis.

In order to detect protein levels of methylated histone H3, HIF-1, and endostatin,

cultured cells were washed with PBS once and then lysed directly in 0.5ml SDS loading

buffer, followed by heat at 99oC for 10 min before western-blot analysis. In addition, mouse

liver was grinded in 0.5% NP-40 buffer, and lysed on ice for 30 min, following heat in SDS

loading buffer at 99oC for 10 min before western-blot analysis. Standard western-blot

21

protocols were adapted.

Quantitative real-time PCR

Total RNA was extracted from mouse livers using Trizol reagent (Invitrogen).

Quantitative real time-PCR was performed following the manufacturer’s instruction using an

Applied Biosystems 7500 Sequence Detection System with SYBR green labeling (Takara).

The primer sequences were listed in Supplemental Table 2. β−actin and Gapdh were used

as endogenous controls.

Immunohistochemical analysis

Mouse livers were fixed in 4% neutral buffered formalin (Shenggong, Shanghai, China),

mounted in paraffin, and then were cut into 5μm thin sections. 5hmC levels were detected by

using a labeled streptavidin-biotin (LSAB) method with commercial kits (Dako Corporation,

Santa Barbara, CA). Briefly, sections were normally deparaffinized and incubated with 3%

H2O2 in PBS for 30 min to eliminate the endogenous peroxidase activity. After microwave

repair for 10 min, the sections were treated with 2N HCl for 15 min at room temperature, and

then wash three times with PBS, followed by incubation with a primary anti-5hmC antibody

(Active Motif; Cat. 39769, dilution at 1:1000) at 37℃ for 1 hour or 4℃ overnight. PBS

(without the primary antibody) was used as a negative control. A horseradish peroxidase

(HRP)-conjugated secondary antibody (Dako Corporation, Santa Barbara, CA) was then

applied and incubated at 37℃ for 1 hour. Sections were developed with a DAB kit and

stopped with water according to the manufacturer’s instructions. Images were captured

22

using a charge-coupled device (CCD) camera and analyzed using Motic Images Advanced

software (version 3.2, Motic China Group CO. Ltd).

Immunofluorescence assay

Cells were fixed with 4% paraformaldehyde (Shenggong) for 1 hour at room

temperature before washing with cold PBS containing 0.4% Triton X-100 for 15 min. For

5hmC staining, permeabilized cells were denatured with 2N HCl for 15 min, neutralized with

100 mM Tris-HCl (pH 8.5) for 10 min, and then washed with PBS for three times. Afterward,

the cells were incubated with blocking buffer (3% bovine serum albumin in PBS containing

0.1% TritonX-100) for 1 hour, followed by incubation for 2 hours at room temperature with

primary antibodies against 5hmC (Active Motif; dilution at 1:5000) or Flag (Sigma-Aldrich,

dilution at 1:1000). Alex Fluor 488 (Red) or 594 (Green)-conjugated secondary antibodies

(Invitrogen) were then applied and incubated at room temperature for 1 hour. Cell nuclei

were stained with DAPI (Invitrogen). Images were captured using Olympus fluorescence

microscope IX18.

Dot-blot assay

The dot-blot assay was performed as described previously (Xu et al, Cancer Cell, 2010)

with some modifications. Briefly, DNA was spotted on a nitrocellulose membrane (Whatman)

which was placed under an ultraviolet lamp for 20 min to crosslink the DNA. Subsequently,

the membrane was blocked with 5% milk in TBS-Tween20 for 1 hour and incubated with the

primary anti-5hmC antibody at 4°C overnight. After incubation with a HRP-conjugated

23

secondary antibody (GeneScript) for 1 hour at room temperature, the membrane was

washed with TBS-Tween 20 for three times and then scanned by a Typhoon scanner (GE

Healthcare). The 5hmC intensity was quantified by Image-Quanta software (GE Healthcare).

Reference

Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, Ito S, Yang C, Wang P, Xiao MT, et al. 2011.

Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent

dioxygenases. Cancer Cell. 19(1):17-30.

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