Molecular Cell, Volume 53

Supplemental Information

Reciprocal Regulation of HIF-1 and LincRNA-p21

Modulates the Warburg Effect Fan Yang, Huafeng Zhang, Yide Mei, and Mian Wu

A

lincRNA-p21 ROR-797- -788 +1 -1518- -1515 +1

479 476 397 394 1316 1313 1

Figure S1

lincRNA-A7

Malat1NEAT1

HOTAIR

-162- -159 -82- -79 +1

-479- -476 -397- -394

-1500- -1497 +1

-1686- -1683-258- -255

-1316- -1313 +1

-661- -664

-1118- -1115 +1

B C20 Glut1B C

25

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opy

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bers

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D E5

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1 2 3 4 5 6

Hypoxia (h) 0 6 12 24 48 72HIF-1αβ-Actin

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ve ofHeLa MCF7 H1299 LO2 IMR90

Hypoxia(h)0

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otal

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ium

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sh-li

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-3

A B

Figure S2

Normoxia + + - -

Digoxin

β ActinHIF-1α

+ + - -Hypoxia - - + + - - + +

- - - - ++++

β-Actin

HIF-1αN H N H N H N H

sh-ctrl + + + +- - - -sh-lincRNA-p21 - - - -+ + + +

sh-HIF-1α - - - - ++ + +

RN

A-p

21-1

RN

A-p

21-2

RN

A-p

21-3

C

1 2 3 4 5 6 7 8β-Actin

1 2 3 4 5 6 7 8

1 2 3 4 5

β-Actin

HIF-1αNormoxia + - - - -

Hypoxia - + + + +

sh-c

trl

sh-li

ncR

sh-li

ncR

sh-li

ncR

Figure S3

sh-ctrl + -

sh-VHLsh-lincRNA-p21 -

- - + - - -- + + - - + +

- + - + - + - +

BA

CHX 0 15 30 60 90 120 0 15 30 60 90120 (min)sh-ctrl sh-lincRNA-p21

IP:

Cβ-ActinVHL

N H N Hsh-ctrl + + - -

sh-VHL - - + +

1 2 3 4 5 6 7 8β-ActinHyp564-HIF-1α

N N N N H H H H

Hyp564-HIF-1α/Actin

β-ActinHyp564-HIF-1α

CHX 0 15 30 60 90 120 0 15 30 60 90120 (min)

10.

950.

570.

450.

180.

14

0.45

0.42

0.23

0.15

0.121

sh-ctrl + - + -sh-lincRNA-p21

N N H H

Input

N N H H+ - + -

- + - + - + - +

IB:Hyp564-HIF-1α

Anti-VHL

IB:VHLIB:β-Actin

1 2 3 4 5 6 7 8

β

1 2 3 4

Figure S4

*

**8

6

4A-p

21 le

vel i

n IP

A C

sh-ctrl + - + -sh-lincRNA-p21

Input

+ - + -- + - + - + - +

IP:

Anti-HIF-2α

B

sh-ctrl + +sh-lincRNA-p21-1 + +sh-lincRNA-p21-2 + +sh-lincRNA-p21-3 + +

ED Input Pulldown

Biotin-lincRNA-p21P ** **

IgG

IgG

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-2α

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L

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0Rel

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A

Antibody

1 2 3 4 5 6 7 8IB:β-Actin

IB:VHLIB:HIF-2α

N N H H N N H H

β-ActinHIF-2α

N H N H N H N H

0.06

0.02

0.54

0.03

0.56

0.05

0.521HIF-2 α /Actin

F Input Pulldown

3xflag-VHL3xflag-VHL(1-186)

3XFl

ag3X

Flag

-VH

L(FL

)3X

Flag

-VH

L(54

-213

)3X

Flag

-VH

L(72

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)3X

Flag

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L(1-

156)

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ag-V

HL(

1-18

6)3X

Flag

3XFl

ag-V

HL(

FL)

3XFl

ag-V

HL(

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13)

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ag-V

HL(

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flag

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76

543210

IgGFlag

bHLHPAS TADHIF-1α(FL)HIF-1α(1-329)HIF-1α(330-530)

bHLHPAS

AD β αVHL(FL)VHL(54-213)VHL(72-213)VHL(1-156)VHL(1-186)

β αβ α

AD βAD β α

G

g ( )3xflag-VHL(1-156)3xflag-VHL(54-213)

3xflag-VHL(72-213)1 2 3 4 5 6 7 8 9 10 11 12

IB:A

nti-f

UVfluorescence lincRNA-p21

Rel 0HIF 1α(330 530)

HIF-1α(531-826) TAD

F1 F2

F1 (1-1028)F2 (1029-1956)

lincRNA-p21FL (1-1956)

F1F2

nti-F

lag

Biotin-lincRNA-p21I J

Input IP

RNA(FL) + +RNA(F1) + +RNA(F2) + +

-PC

R

FL

H

Inpu

t

IP: a

n

lincRNA-p21 RNA + + + + + +

3xFlag - -- -3xFlag-HuR - + - - + -3xFlag-VHL - - + - - +

RT-PCR

+ +

66kD GST-HuRGST-VHL

45kD

Mar

ker

GS

T

GS

T-V

HL

GS

T-H

uR

GS

TG

ST-

HuR

GS

T-V

HL

Input Pulldowm

M

WB

RT- F1/F2

1 2 3 4 5 6 7 8 9 101112

3xflag-VHLIgG Lc

K Input Pulldown

0) 6) 0) 6)

Biotin-lincRNA-p21L

3Xflag-VHL3Xflag-HuR

1 2 3

WB: anti-Flag Coomassie staining

IB:Anti-GST

35kD

Input IP

RNA(FL) + +RNA(F1) + +RNA(F2) + +

T-P

CR

FLF1/F2

5

4

3

2RN

A-p

21 le

vel i

n IP IgG

Flag ** *** *

3xflag-HIF-1α

3xFl

ag3x

Flag

-HIF

-1α(

FL)

3xFl

ag-H

IF-1α(

1-32

9)3x

Flag

-HIF

-1α(

330-

530

3xFl

ag-H

IF-1α(

531-

826

3xFl

ag3x

Flag

-HIF

-1α(

FL)

3xFl

ag-H

IF-1α(

1-32

9)3x

Flag

-HIF

-1α(

330-

530

3xFl

ag-H

IF-1α(

531-

826

ag

WB

RT F1/F2

HIF-1α

1 2 3 4 5 6 7 8 9 10 1112

2

1

0

Rel

ativ

e lin

cR 3xflag-HIF-1α(531-826)3xflag-HIF-1α(1-329)3xflag-HIF-1α(330-530)

1 2 3 4 5 6 7 8 9 10

IB:A

nti-f

l

UVfluorescence lincRNA-p21

*

Figure S5

A B

***25 **8

DOX-DOX+

C

c

elat

ive

mlin

cRN

A-p

21 le

vel

***

5

10

15

20DOX 0 μgDOX 0.5 μgDOX 1 μg

Rel

ativ

e lin

cRN

A-p

21 le

vel

6

4

2

DOX+

β-Actin

lincRNA-p21

Normoxia + - + -Hypoxia - -+ +

Nuc

lear

Cyt

opla

smic

RT-PCR

Re

sh-ctrl sh-mlincRNA-p210

R0

sh-c

trl

sh-li

ncR

NA

-p21

-2

sh-li

ncR

NA

-p21

-3

U6 snRNA

β Actin

D

Human lincRNA-p21

(partial sequence)Chr6: 36635073 36634046 36633249 36632321

(1956 nt)(Exon 1) (Intron 1) (Exon 2)

D

Supplemental Figure Legends

Figure S1, related to Figure 1

(A) Schematic representation of consensus hypoxia-responsive binding sites (CGTG)

(Red stars) in the indicated lncRNA promoter region. Arrowheads indicate the

orientation of transcription.

(B) HeLa cells were cultured under hypoxic (1% O2) conditions for the indicated

periods of time. Total RNA was subjected to real-time RT-PCR analysis. The data are

shown as mean ±SD of three independent experiments. HIF-1α expression upon

hypoxia treatment was also analyzed by Western blotting.

(C) The copy numbers of lincRNA-p21 transcript per cell in the indicated tumor cells

and non-transformed cells were quantified using a quantitative real-time RT-PCR

assay. Data shown are mean ± SD (n=3).

(D) HeLa cells were infected with lentiviruses expressing either control or three

different sets of lincRNA-p21 shRNAs. Twenty-four hours after infection, total RNA

was subjected to real-time RT-PCR analysis. Shown data are mean±SD (n=3).

(E) 2×105 HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were

cultured under normoxic or hypoxic conditions for 24 hrs. Cell numbers were then

counted. The data are shown as mean±SD of three independent experiments.

(F) HeLa cells expressing either control shRNA, lincRNA-p21-1 shRNA or

lincRNA-p21-2 shRNA were cultured under normoxic or hypoxic conditions for 24

hrs. Acidification of the culture medium was evaluated by visually inspecting the

color of the medium.

(G) HeLa cells expressing either control shRNA or the indicated lincRNA-p21

shRNAs were cultured under normoxic or hypoxic conditions for 24 hrs. pH value of

the culture medium was then measured. The data are shown as mean±SD of three

independent experiments.

Figure S2, related to Figure 2

(A) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were

co-transfected with the HIF-1α-responsive element (HRE) reporter construct and

Renilla luciferase plasmid. Twelve hours later, cells were cultured under normoxic or

hypoxic conditions in the presence or absence of Digoxin for additional 24 hrs. Hif-1α

expression was then examined by Western blot analysis.

(B) HeLa cells expressing control shRNA, lincRNA-p21 shRNA, HIF-1α shRNA or

both lincRNA-p21 and HIF-1α shRNAs were co-transfected with the

HIF-1α-responsive element (HRE) reporter construct and Renilla luciferase plasmid.

Twelve hours later, cells were cultured under normoxic or hypoxic conditions for

additional 24 hrs before Hif-1α expression was determined by Western blot analysis.

(C) HeLa cells expressing control shRNA, lincRNA-p21-1 shRNA, lincRNA-p21-2

shRNA or lincRNA-p21-3 shRNA were cultured under normoxic or hypoxic

conditions for 24 hrs. Cell lysates were then analyzed by Western blotting.

Figure S3, related to Figure 3

(A) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were

transfected with p3xflag-myc-cmv-24-HIF-1α plasmid. Twelve hours later, cells were

cultured under hypoxic conditions for additional 24 hrs. Cells were then treated with

CHX (25 μg/ml) for the indicated periods of time before cells were harvested for

Western blot analysis.

(B) HeLa cells expressing control shRNA, lincRNA-p21 shRNA, VHL shRNA or

both VHL and lincRNA-p21 shRNAs were cultured under normoxic or hypoxic

conditions for 24 hrs. Cell lysates were then analyzed by Western blotting. Successful

knockdown of VHL was also confirmed by Western blot analysis.

(C) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were

cultured under normoxic or hypoxic conditions in the presence of MG132 (5 μM) for

24 hrs. Cell lysates were immunoprecipitated with anti-VHL antibody. The

immunoprecipitates and input were then analyzed by Western blotting with the

indicated antibodies.

Figure S4, related to Figure 4

(A) HeLa cells were cultured under normoxic or hypoxic conditions for 24 hrs before

IP-real-time RT-PCR assays were carried out. Shown data are mean±SD (n=3).

(B) HeLa cells expressing either control shRNA or the indicated lincRNA-p21 shRNA

were cultured under normoxic or hypoxic conditions for 24 hrs. Cell lysates were then

analyzed by Western blotting with the indicated antibodies.

(C) HeLa cells expressing either control shRNA or lincRNA-p21 shRNA were

cultured under normoxic or hypoxic conditions in the presence of MG132 (5 μM) for

24 hrs. Cell lysates were immunoprecipitated with anti-HIF-2α antibody. The

immunoprecipitates and input were analyzed by Western blotting.

(D) Schematic illustration of full length VHL and HIF-1α and their deletion mutants

used in this study.

(E) HeLa cells were transfected with p3×flag-myc-cmv-24-VHL or its deletion

mutants as indicated. Twelve hours after transfection, cells were cultured under

hypoxic conditions for additional 24 h. Cell lysates were immunoprecipitated with

anti-Flag antibody or an isotype-matched IgG before lincRNA-p21 present in the

immunoprecipitates was examined by real-time RT-PCR. Data shown are mean ± SD

(n=3).

(F) In vitro transcribed biotin labeled lincRNA-p21 was incubated with lysates from

HEK293T cells expressing full length VHL or the indicated VHL deletion mutants for

3 hrs. A biotin pull-down assay was then carried out.

(G) Schematic illustration of full length lincRNA-p21 and its mutants used in this

study.

(H) HeLa cells were transfected with p3×flag-myc-cmv-24-VHL. Twenty-four hours

after transfection, cell lysates were incubated with in vitro transcribed RNAs

corresponding to different fragments of lincRNA-p21, followed by

immunoprecipitation with anti-Flag antibody. RNAs present in the input and

immunoprecipitates were analyzed by RT-PCR.

(I) In vitro transcribed lincRNA-p21 was incubated with purified flag-tagged HuR or

VHL bound with M2 beads. After incubation, beads were washed and bead-bound

RNA was eluted as templates for RT-PCR analysis. HuR was used as a positive

control in this assay because it has been shown to bind lincRNA-p21.

(J) In vitro transcribed biotin labeled lincRNA-p21 was incubated with purified

recombinant GST, GST-HuR or GST-VHL proteins bound with glutathione agarose

beads. After incubation, beads were washed and beads-bound proteins were eluted,

followed by Western blot analysis with anti-GST antibody. The input proteins were

also visualized by Coomassie blue staining. HuR was used as a positive control in this

assay because it has been shown to bind lincRNA-p21.

(K) HeLa cells were transfected with pcDNA-V5/His-HIF-1α. Twenty-four hours

after transfection, cell lysates were incubated with in vitro transcribed RNAs

corresponding to different fragments of lincRNA-p21, followed by

immunoprecipitation with anti-HIF-1α antibody. RNAs present in the input and

immunoprecipitates were analyzed by RT-PCR.

(L) HeLa cells were transfected with p3×flag-myc-cmv-24-HIF-1α or its various

deletion mutants as indicated. Twelve hours after transfection, cells were cultured

under hypoxic conditions for additional 24 h. Cell lysates were immunoprecipitated

with anti-Flag antibody or an isotype-matched IgG. LincRNA-p21 present in the

immunoprecipitates was examined by real-time RT-PCR. Data shown are mean ± SD

(n=3).

(M) HEK293T cells were transfected with the indicated plasmids expressing full

length HIF-1α or its various deletion mutants as indicated. Cell lysates were then

incubated with in vitro transcribed biotin labeled lincRNA-p21 for 3 h before a biotin

pull-down assay was carried out. * indicates non-specific band.

Figure S5, related to Figure 6

(A) MEF cells expressing control shRNA or mlincRNA-p21 shRNA were treated with

the indicated concentration of doxorubicin for 24 hrs. Cell lysates were then subjected

to RNA extraction, followed by real-time RT-PCR analysis. Data are represented as

mean±SD (n=3).

(B) HeLa cells expressing control shRNA, lincRNA-p21-2 shRNA or lincRNA-p21-3

shRNA were treated with 1 μg/ml doxorubicin for 24 hrs before real-time RT-PCR

analysis were performed. Data are represented as mean±SD ( n=3).

(C) HeLa cells were cultured under normoxic (20% O2) or hypoxic (1% O2)

conditions for 24 hrs. The cytosolic and nuclear fractions were then prepared. The

distribution of lincRNA-p21 was detected by RT-PCR. U6 snRNA and β-Actin

mRNA were useded as a nuclear marker and a cytosolic marker, respectively.

(D) Genomic localization of human lincRNA-p21 partial sequence.

Primers used in qRT-PCR assays HIF-1α Fw:5'GAAGACATCGCGGGGAC3'

Rev:5'TGGCTGCATCTCGAGACTTT3' β-Actin Fw: 5'GACCTGACTGACTACCTCATGAAGAT3’

Rev:5'GTCACACTTCATGATGGAGTTGAAGG3’ human lincRNA-p21 Fw:5'GGGTGGCTCACTCTTCTGGC3'

Rev:5'TGGCCTTGCCCGGGCTTGTC3' mouse lincRNA-p21 Fw:5'GGAGTCTCATGCTCAGAGAAGAA3'

Rev:5'CCCTGACAGACAAGTACCCTCT3' LDHA Fw:5'ACCCAGTTTCCACCATGATT3'

Rev:5'CCCAAAATGCAAGGAACACT3' GLUT1 Fw:5'CGGGCCAAGAGTGTGCTAAA3’

Rev:5'TGACGATACCGGAGCCAATG3’ MALAT1 Fw:5'AATGTTAAGAGAAGCCCAGGG3'

Rev:5'AAGGTCAAGAGAAGTGTCAGC3' NEAT1 Fw:5'ACTTGATAACACCCACACCC3'

Rev"5'ACAGAGTCACCAGTTTTCCG3' HOTAIR Fw:5'GGGGCTTCCTTGCTCTTCTTATC3'

Rev:5'GGTAGAAAAAGCAACCACGAAGC3' LncRNA-A7 Fw:5'CCGTTGGCTCCACAAACCT3'

Rev:5'CAGTGACAGTAGCAGGCATCCT3' ROR Fw:5'CTTGATGGCATTGTCGCTAA3'

Rev:5'TCCAGTGGCTGTGCTAGATG3' Primers used in IP-RT-PCR assays LincRNA-p21 (FL) 5' GGCCGGCAGAGCTCCCGCTGG 3'

5' TACAGGTGCCCACCACTACG 3' LincRNA-p21 (F1) 5' GGCCGGCAGAGCTCCCGCTGG 3'

5' CACAGCTAGCAGGACCTAC 3' LincRNA-p21 (F2) 5' CAGAAACAAGACCTACTGAGG 3'

5' TACAGGTGCCCACCACTACG 3' Oligonucleotide sequence of shRNAs sh-HIF-1α#1 5' CCGCTGGAGACACAATCATAT 3' sh-HIF-1α#2 5' CCAGTTATGATTGTGAAGTTA 3' sh-lincRNA-p21#1 5' GCCACCCACTCACCACTGG 3' sh-lincRNA-p21#2 5' GGAGGACACAGGAGAGGCA 3' sh-lincRNA-p21#3 5' GCATTGTTGCATCATCATG 3' sh-p53 5' GACTCCAGTGGTAATCTAC 3' sh-HIF-2α#1 5' AGGTGGAGCTAACAGGACATA 3' sh-HIF-2α#2 5' CGACCTGAAGATTGAAGTGAT 3' Primers for in vitro transcription The T7 RNA polymerase sequence(T7) was 5' CCAAGCTTCTAATACGACTCACTATAGGGAGA 3'LincRNA-p21 (FL) 5' (T7)GGCCGGCAGAGCTCCCGCTGG 3'

5' TACAGGTGCCCACCACTACG 3'

Table S1

LincRNA-p21 (F1) 5' (T7)GGCCGGCAGAGCTCCCGCTGG 3' 5' CACAGCTAGCAGGACCTAC 3'

LincRNA-p21 (F2) 5' (T7)CAGAAACAAGACCTACTGAGG 3' 5' TACAGGTGCCCACCACTACG 3'

β-Tubulin CDS 5' (T7)ATGAGGGAAATCGTGCACATCC 3' 5' TTAGGCCTCTTCGGCCTCC 3'

Primers used in ChIP assays LDHA HRE Fw: 5' TTGGAGGGCAGCACCTTACTTAGA 3’

Rev: 5' GCCTTAAGTGGAACAGCTATGCTGAC 3’ P1 Fw: 5' GCTGCCCAGACTGGAGTGC 3'

Rev: 5'TGGGGCCACACACTGCCT 3' P2 Fw: 5' CTCAAGTGATCCGCCTGCCTC 3'

Rev: 5' CATCCTGGACCCCAGGCTG 3' 18S rRNA Fw: 5' CGGCGACGACCCATTCGAAC 3’

Rev: 5' GAATCGAACCCTGATTCCCCGTC 3’