ulementary inormation - nature · of bodipy fl-pip 3 (donor) in the presence of alexa-555-strep...

S U P P L E M E N TA RY I N F O R M AT I O N

WWW.NATURE.COM/NATURECELLBIOLOGY 1

DOI: 10.1038/ncb3473

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

In the format provided by the authors and unedited.

Lin, et al, 2016 Supplementary Figure 1

b

Fo

ld C

ha

ng

e

0

10

20

30

40

50

LINC01

139

RP11

-433

A10

.1

SNHG6

XLO

C_0

0238

4

LINC00

607

RP5-

858B

6.3

SNHG9

RP11

-383

G10

.5

SNHG3

a

010203040

60708090

50

LINC01

139

RP11

-433

A10

.1

SNHG6

XLO

C_0

0238

4

LINC00

607

RP5-

858B

6.3

SNHG9

RP11

-383

G10

.5

SNHG3

Lip

id E

nrich

me

nt

Fa

cto

r (L

/T%

) Normal BT

TNBC

E

c

0

0.05

0.1

0.15

0.2

0.25

0.3

% o

f In

pu

t

Lipid-coatedBeads

CTL

PAPC

PE P

S

Ptd

Ins

PI(3

)P

PI(4

)P

PI(5

)P

PI(3

,4)P

2

PI(3

,5)P

2

PI(4

,5)P

2

PI(3

,4,5

)P3

Cer

amide

LBPA

Sph

ingo

mye

lin

Sph

ingo

sine

S1PLP

A

Car

diolipin

PEth

+RT-RT

Lipid-coated Beads Pull-down

RNA GAPDH

0

0.05

0.1

0.15

0.2

0.25

0.3

Lipid-coatedBeads

CTL

% o

f In

pu

t

PAPC

PE P

S

Ptd

Ins

PI(3

)P

PI(4

)P

PI(5

)P

PI(3

,4)P

2

PI(3

,5)P

2

PI(4

,5)P

2

PI(3

,4,5

)P3

Cer

amide

LBPA

Sph

ingo

mye

lin

Sph

ingo

sine

S1P LP

A

Car

diolipin

PEth

+RT-RT

Lipid-coated Beads Pull-down

RNA LINK-A

*

*

d

e

0

2.0

4.0

6.0

Flu

ore

sce

nce

In

ten

sity (

x1

0)

4

500 510 520 530 540 550

Wavelength (nm)

BODIPY FL-PIP3

+ Alexa-555-Strep

BODIPY FL-PIP3

+ Alexa-555-Strep-Biotin- (400 nM)RP11-383G10.5

Alp

ha

Sig

na

l (x

10

00

)

0

10

20

35

5

15

25

30

-11 -10 -9 -7 -6 -5 -4

Unlabeled LINK-A

Full Length Log(M)

-8 -3

142.3 8.4 nM=Kd

DIG- +Biotin-PI(3,4,5)PLINK-A 3

10

20

35

5

15

25

30

0-11 -10 -9 -7 -6 -5 -4

Unlabeled LINK-A

(PC-binding motif) Log(M)

-8 -3

=IC50 N.D.

10

20

35

5

15

25

30

0-11 -10 -9 -7 -6 -5 -4

Unlabeled LINK-A

(PIP -binding motif) Log(M)3

-8 -3

4.1 0.6 M�=IC50

g

Emission520-620nm

Excitation680nm 1/2 O2

DIG-LINK-A

BIO-PIP3

StreptavidinAcceptor Beads

Anti-DIGDonor Beads

f

Supplementary Figure 1 Characterization of lipid-lncRNA interaction. (a) Lipid binding affinity (Enrichment, lipid/total, L/T %) of TNBC-upregulated lncRNAs were calculated using normalized density. (b) Fold change of lncRNAs expression in tumor tissues compared to normal breast tissue. (c and d) Lipid-coated beads pull-down followed by RT-qPCR detection of LINK-A-lipid (c) or GAPDH-lipid (d) interactions. (e) Fluorescence spectra of BODIPY FL-PIP3 (donor) in the presence of Alexa-555-Strep alone (black line) or with Alexa-555-Strep-biotin-RP11.38310.5 (red line). (f)

Graphic illustration of Alpha assay using DIG-LINK-A and Biotin-PIP3. (g) Competition binding Alpha assay to determine Kd for interaction between Biotin-PIP3 and DIG-LINK-A, in the presence of unlabeled full-length LINK-A (left), PC-binding motif (middle) or PIP3-binding motif (right) titrated from 0.4 mM to 0.05 nM (mean ± s.e.m. were derived from n=3 independent experiments). For c and d, mean ± s.e.m. were derived from n=3 independent experiments (*p<0.05, two-tailed paired Student’s t-test).

http://dx.doi.org/10.1038/ncb3473





0

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0 10 20 304050 60 70 80

Distance (nm)

Inte

nsity

Intensity Ch1

Intensity Ch2

90100

DO

PC

YO

YO

-1O

ve

rla

p

50nm 0

50

100

150

200

250

0 20 40 60 80 100120140160

Distance (nm)

Inte

nsity

Intensity Ch1

Intensity Ch2

100nm

DO

PC

BC

AR

4O

ve

rla

p

b

Inte

nsity

Ch2

Intensity Ch1

0

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250

0 50 100 150 200 250

0 50 100 150 200 250

Inte

nsity C

h2

0

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250

0 50 100 150 200 250

0 50 100 150 200 250

Intensity Ch1

Overlap Coefficient 0.90

Distance (nm)

0

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0 10 20 30 4050 60 70 80

Inte

nsity

Intensity Ch1

Intensity Ch2

0

50

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250

0 10 203040 5060 7080

Distance (nm)

Inte

nsity

Intensity Ch1

Intensity Ch2

90

DO

PC

LIN

K-A

Ove

rla

p

50nm

DO

PC

Lin

c-1

31

Ove

rla

p

50nm

Inte

nsity C

h2

0

50

100

150

200

250

0 50 100 150 200 250

0 50 100 150 200 250

Intensity Ch1

Overlap Coefficient 0.00

LINK-A Linc-131a

c

Permeable IPn DM

SO

DM

SO

Ins(1

,4,5

,6)P

4

Ins(1

,,4

,5,6

)P5

3

DM

SO

DM

SO

Ins(1

,4,5

,6)P

4

Ins(1

,,4

,5,6

)P5

3

DM

SO

DM

SO

Ins(1

,4,5

,6)P

4

Ins(1

,,4

,5,6

)P5

3

GST-MS2 - + - + - +

MS2-LINK-A

Input IP:IgG IP:PI3K

p110�

MDA-MB-231

IB:PI3K p110�

Input IP:IgG

MDA-MB-231

IP:PI3K

p110�

GST-MS2 - +

FL

- + - + - + - + - +

MS2-LINK-A �P

IP3

FL

�P

IP3

FL

�P

IP3IB:PI3K p110�

e f

h i

d

g

0

5

10

15

20

25

30

35

Re

lative

Exp

ressio

nL

eve

l o

f M

S2

-LIN

K-A

GST-MS2 - - + + +

MS2-LINK-A

Bla

nk

***� �PI3K p110 �IgG

0

2

4

6

% o

f P

IPto

PIP

23

Convers

ion

DM

SO

DM

SO

Ins

(1,4

,5,6

)P4

Ins

(1,

,4,5

,6)P

53Permeable IPn

GST-MS2 +-

MS2-LINK-A

n.s.n.s.

n.s.

� �PI3K p110 �IgG

0

2

4

6

% o

f P

IPto

PIP

23

Convers

ion

GST-MS2 - + - +

MS2-LINK-A FL �PIP3

n.s.n.s.

n.s.

0

5

10

15

20

25

30

Re

lative

Exp

ressio

nL

eve

l o

f M

S2

-LIN

K-A

GST-MS2 -

FL

- + - +

MS2-LINK-A

�P

IP3

Bla

nk

40

35

***

Ove

rla

p C

oe

f ficie

nt

LINK-A

BCAR4

H19

Linc

-131

***

0.0

0.5

1.0

1.5 LINK-A

BCAR4

H19

Linc-131

Supplementary Figure 2 Determination of LINK-A-PIP3 and LINK-A-Ins (1,3,4,5)P4 interactions by giant unilamellar vesicles and MS2-TRAP. (a and b) Fluorescence imaging of interaction between DOPC lipid vesicles (visualized by Nile Red) and indicated lncRNA (visualized by YOYO-1) (a) or YOYO-1 dye only (b). Left panel: Representative images. Scale bars, 50 nm. Middle and right panels: fluorescence intensities along the diagonal bars (middle panel) and intensity correlation between two channels (right panel) were shown. (c) Overlap coefficient between channel 1 (Nile Red) and channel 2 (YOYO-1) was calculated based on the number of giant lipid vesicles, LINK-A (n=34 lipid vesicles), BCAR4 (n=19 lipid vesicles), Lnc-131 (n=21 lipid vesicles), and H19 (n=7 lipid vesicles) (median, one-way ANOVA, ***p<0.001). (d and g) Relative expression level of MS2-tagged full-length LINK-A (d) or ΔPIP3

deletion mutant (g) in MDA-MB-231 cells was detected by RT-qPCR. (e and h) ELISA assay detecting the conversion of PIP2 to PIP3 by immunoprecipitated PI3K p110α from cells pretreated with DMSO, PI(1,4,5,6)P4 or PI(1,3,4,5,6)P5 (100 µM, 2 hrs) (e) or transfected with MS2-tagged full-length LINK-A or ΔPIP3 deletion mutant (h). (f and i) Immunoblotting detection of immunoprecipitated PI3K p110α from cells pretreated with DMSO, PI(1,4,5,6)P4 or PI(1,3,4,5,6)P5 (100 µM, 2 hrs) (f) or transfected with MS2-tagged full-length LINK-A or ΔPIP3 deletion mutant (i). For d, e, g and h, mean ± s.e.m. were derived from n=3 independent experiments (n.s. p>0.05 and ***p<0.001, two-tailed paired Student’s t-test). Statistics source data for a are in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.





0

10

30

20

40

Alp

ha

Sig

na

l (x

10

00

)

0 0.2 0.8 1

Biotin-Ins(1,3,4,5)P ( M)4 �

0.4 0.6

DIG- (FL)LINK-A

196.5 13.8 nMKd =

0

10

30

20

40

DIG- ( PC)LINK-A �

0 0.2 0.8 1

Biotin-Ins(1,3,4,5)P ( M)4 �

0.4 0.6

221.7 22.6 nMKd =

0

10

30

20

40 0.2 M�

0.1 M�

0.05 M�

0 0.2 0.8 1

Biotin-Ins(1,3,4,5)P ( M)4 �

0.4 0.6

DIG- ( PIP )LINK-A � 3

Kd = N.D.

-11 -10 -9 -7 -6 -5 -4

Unlabeled Ins(1,3,4,5)P ] Log(M)4

-8 -30

10

20

15

25

Alp

ha

Sig

na

l (x

10

00

)

5

DIG- +Biotin-Ins(1,3,4,5)PLINK-A 4

210.9 9.3 nMKd =

a

b**

0

0.04

0.08

0.12

0.16

% o

f In

pu

t0

0.04

0.08

0.12

0.16

0.2

MDA-MB-468

�PIP3

IgG

- + - +

LINK-A GAPDH

MDA-MB-231

LY294002 - + - +

LINK-A GAPDHRNA

* **

c d

LY294002 - + - +

MD

A-M

B-

23

1

MD

A-M

B-

46

8IB:AKT

IB:p-AKT(Ser473)

Supplementary Figure 3 Determination of LINK-A-PIP3 and LINK-A-Ins (1,3,4,5)P4 interactions by RIP and Alpha assay respectively. (a) Saturation curve used to determine Kd of the interactions between Biotin-Ins (1,3,4,5)P4 and Digoxigenin-labeled full-length LINK-A (left panel), ΔPC LINK-A (middle panel), or ΔPIP3 LINK-A (right panel) in Alpha format (mean ± s.e.m. were derived from n=3 independent experiments). (b) Competition binding assay to determine Kd for the interactions between biotin- Ins(1,3,4,5)P4 and Digoxigenin-labeled LINK-A in the

presence of unlabeled Ins(1,3,4,5)P4 as competitor (mean ± s.e.m. were derived from n=3 independent experiments). (c and d) Immunoblotting detection (c) or RIP-qPCR detection of indicated RNAs retrieved by PIP3-specific antibody (d) in MDA-MB-231 cells treated with DMSO or LY294002. For a, b and d, mean ± s.e.m. were derived from n=3 independent experiments (*p<0.05 and ***p<0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.





0

20

40

60

80

100

120

Mem

.

Cyto.

***

Ab

so

lute

Co

py N

um

be

ro

fp

er

Ce

llL

INK

-A

140

160

Mem

.

Cyto.

IB: Na/K ATPase

IB: -Tubulin�

a b c

0

20

40

60

80

100

120

% o

f E

xp

ressio

n L

eve

l

MembraneCytosol

LINK-A B2M

******

e

d

0

0.2

0.4

0.6

0.8

1.0

1.2

LIN

K-A

Re

lative

Exp

ressio

n L

eve

l

Scr

LNA#1

LNA#2

LNA#3

LNA#4

LNA#5

*

0

500

1000

2000

1500

LNA Scr LINK-A #5

Bla

nk

Ve

cto

r

Bla

nk

Ve

cto

r

LIN

K-A FL

LIN

K-A

PIP

�3

LIN

K-A PC

�

LIN

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Exp

ressio

nR

ela

tive

Fo

ld C

ha

ng

e

- EGF

+ EGF*

0

0.05

0.1

0.15

0.2

0.25

Re

lative

Kin

ase

Activity (

OD

45

0n

m)

In Vitro AKT Kinase Assay

** **

n.s.

n.s.n.s.

1 2 3 4 5 6 7 8

no AKTAKT

AKT+LINK-A

AKT Ctl PolyPIPosomes+

AKT PIP PolyPIPosomes+ 3

AKT PIP PolyPIPosomes++ LINK-A3

AKT PIP PolyPIPosomes+ PIP+ LINK-A,3 3�

AKT PIP PolyPIPosomes+ PC+ LINK-A,3 �

1

2

3

4

5

6

7

8

g

0

0.2

0.4

0.6

0.8

1.0

% o

f In

pu

t

EGF +- +- +- +- +-

LNA Scr

LINK-A

Vecto

r

Vecto

r FL�PC

�PIP

3

LINK-A #5

+- +- +- +- +-

Vecto

r

Vecto

r FL�PC

�PIP

3

�PIP3

IgG

RIP

Scr LINK-A #5

LINK-A RP11-383G10.5

n.s.

**

*

n.s.

f

Supplementary Figure 4 Characterization of LINK-A subcellular localization, knockdown efficiency and effect on AKT kinase activity in vitro. (a) Immunoblotting of membrane and cytoplasmic fractionations from MDA-MB-231treated with EGF (10 ng/ml) for 30 minutes using indicated antibodies. (b) RT-qPCR detection of LINK-A expression in membrane and cytoplasmic fractionations, B2M was used as a cytoplasmic RNA control. (c) Determination of copy number of LINK-A in MDA-MB-231 cells. (d) RT-qPCR analyses of LINK-A knockdown efficiency in MDA-MB-231 cells transfected with indicated LNAs. (e) RT-qPCR analyses of LINK-A expression level in MDA-MB-231 cells transfected with LNA against LINK-A followed

by overexpression of indicated rescue plasmids and EGF stimulation. (f) RIP-qPCR detection of indicated RNAs retrieved by PIP3-specific antibody in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of indicated rescue plasmids with or without EGF treatment. (g) Quantification of AKT kinase activity in the presence of control or PIP3 polyPIPosomes, with or without full-length LINK-A, ΔPIP3 or ΔPC deletion transcripts. For b, c, d, e, f and g, mean ± s.e.m. were derived from n=3 independent experiments (n.s. p>0.05, *p<0.05, **p<0.01 and ***p<0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.





a b

BRK Binding #1nt. 471-550

BRK Binding #2nt. 771-850

LRRK2 Bindingnt. 1251-1330

PIP3 binding loop

PC Bindingnt. 241-300

d

PIP3-binding loop

0

1

2

MS

2-

Re

trie

va

lL

INK

-A

(% o

f In

pu

t)

0.5

1.5

�GST-tag �IgGMS2-TRAP

GST-MS2BP

- +

MS2-LINK-A

WT

EGF - + - + - + - + - +

MS2 �PIP3 3C 4A 6A

MDA-MB-231

n.s.

WT CAGGGU GCUCUGAGACUC

CAGGGU GCUCUGAC

CAGGGU GCUCUGCGACUC

CAGGGU GCUCUGA ACUCU

CAGGGU GCUCUGAG CUCC

CAGGGU GCUCUGAGA UCA

CAGGGU GCUCUGAGAC CG

CAGGGU GCUCUGAGACUA

DIG- (nt. 1100-1117)LINK-A

�2-5

1A

2G

3A

4C

5U

6C

C

U

C

A

G

A

c

Ma

rke

r0.5

1

1.5

22.5

34569

(kb

)

WT 3C 4A 6A

BiotinylatedLINK-A

e

Supplementary Figure 5 Secondary structural modeling of LINK-A-protein and –lipid interactions. (a) Graphic illustration of predicted LINK-A secondary structure and the stem loops corresponding to protein (black dot line circle) and lipid (red dot line circle) binding. (b) Secondary structure of LINK-A (nt. 1,081-1,140) harboring a stem-loop corresponding to PIP3 binding (red dot line circle). (c) List of DIG-labeled wild-type LINK-A and mutant oligonucleotides used in Alpha assay. (d)

RNA agarose gel of in vitro transcribed biotinylated LINK-A (wild-type and single nucleotide mutants). (e) Relative retrieval level of MS2-tagged full-length LINK-A or indicated mutants in MS2-TRAP assay detected by RT-qPCR. For e, mean ± s.e.m. were derived from n=3 independent experiments (n.s. p>0.05, two-tailed paired Student’s t-test). Unprocessed original scans of all blots and gels with size marker are shown in Supplementary Fig. 9.





0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

D1 D2 D3 D4 D5D0

**Scr LNA

LINK-A LNA + Blank vector

LINK-A LINK-ALNA + FL

LINK-A LINK-ALNA + PIP3�

Ab

so

rba

nce

(5

90

nm

)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

D1 D2 D3 D4 D5D0

**LINK-A PIP� 3

Blank Vector

LINK-A FL

Ab

so

rba

nce

(5

90

nm

) 0.9

IB:p-GSK-3�

(Ser9)

MCF-10A

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LINK-A

FL

LINK-A

�PIP3

EGF

IB:AKT

IB:p-AKT(Ser473)

IB:p-AKT(Thr308)

IB:GSK-3�

0 15 30 60 min0 15 30 60 0 15 30 60

a b c d

e f

1 2 3 4 5 6Log copy number LINK-A

12

16

20

24

32

28

Me

an

CT

y=-3.363x+34.82R =0.998

2

g

0

100

200

150

50

Ve

c.

Ve

c.

FL

PIP

3�

Co

py N

um

be

ro

f/C

ell

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K-A

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**

LINK-A

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40

80

120

160

Co

py N

um

be

ro

f/C

ell

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K-A

MCF-10A

Ve

c.

FL

�P

IP3

MD

A-

MB

-23

1

***

LINK-A

jh i

0

50

100

150

Co

py n

um

be

rL

INK

-A

WTRP11-

383G10

3C 4A 6A

LINK-A

Intracellular RNA delivery

-EGF +EGFDLD-1 PIK3CA +/+

***

0

50

100

150

Co

py n

um

be

rL

INK

-A

200-EGF +EGF

DLD-1 PIK3CA +/-

RNA

PIP3 + +

WT

+ +

Intracellular delivery

3C 4A 6ARP11-

383G10 LINK-A

+

***

RNA

PIP3 + +

WT

+ +

Intracellular delivery

3C 4A 6ARP11-

383G10 LINK-A

+

PIP

(pm

ol)/5

x1

03

6ce

ll

0

0.5

1

1.5

2

2.5 -EGF +EGF

DLD-1 PIK3CA +/-

n.s.

PIP ELISA3

LNA Scr LINK-A #5

Bla

nk

Ve

cto

r

Bla

nk

Ve

cto

r

LIN

K-A

FL

LIN

K-A

PIP

3�

EGF - + - + - + - +

IB:AKT

IB:p-AKT(Ser473)

IB:p-AKT(Thr308)

IB:p-GSK-3�

(Ser9)

MDA-MB-231

IB:GSK-3�

Supplementary Figure 6 Determination of LINK-A copy number and characterization of functional sufficiency of LINK-A copy number. (a) Generation of a standard curve for calculating LINK-A copy number. In vitro transcribed LINK-A (a range of amounts from 10 copies to 106 copies) was used to generate cDNAs and generated cDNAs were used in real-time PCR. The resultant CT values decreased linearly with increasing LINK-A copy number, indicating sensitive detection from a wide range of template amounts. (b-d) Determination of LINK-A copy number (b), AKT/GSK-3β activation (c) and cell proliferation (d) in MDA-MB-231 cells transfected with LNA against LINK-A followed by overexpression of indicated rescue plasmids and EGF stimulation. (e-g) LINK-A copy number (e), AKT/GSK-3β activation (f) and cell proliferation (g) were determined by RT-

qPCR, immunoblotting and cell proliferation assay in MCF-10A cells stably expressing full-length LINK-A or ΔPIP3 deletion mutant, with or without EGF stimulation. (h and i) RT-qPCR determination of LINK-A copy number (h) and quantification of cellular PIP3 in DLD-1 PIK3CA+/-cells delivered with PIP3 and indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. (j) RT-qPCR determination of LINK-A copy number in DLD-1 PIK3CA+/+ cells delivered with indicated LINK-A single nucleotide mutated transcripts with or without EGF stimulation. For b, d, e, g, h, i and j, mean ± s.e.m. were derived from n=3 independent experiments (n.s. p>0.05, **p<0.01 and ***p<0.001, two-tailed paired Student’s t-test). Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.





100

200

300400500

1000

(bp

)

Ma

rke

r

1 2 3 4 5 6 7 WT

WT

LINK-A PIP BM3

-/-

a

b

Target sequenceof LINK-A

GATTATAATTATTTGTCTAAGGTGGAGATTACCAGGGTAGACTCGCTCTGGCCGTATGTAATGATGTCTGTGGCTACATAACCTGCC

GCCGTATGTAATGATGTCTGTGTCTAAGGTGGAGATTACCgRNA#2 gRNA#1

5’- -3’

cATGCTAGATTATAATTATTTGTCTAAGGTGGAGATT------------------------------------TCTGTGGCTACATAACCTGCCAACACTTAG

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

ATGCTAGATTATAATTATTTGTCTAAGGTGGAGATTACCAGGGTAGACTCGCTCTGGCCGTATGTAATGATGTCTGTGGCTACATAACCTGCCAACACTTAG

LINK-A PIP BM3

-/-

clone #3

LINK-A PIP BM3

+/+

PIP binding motif (nt. 1081-1140)3

36 nucleotides deletion

ATGCTAGATTATAATTATTTGTCTAAGGTGGAGA ------------------------------------TCTGTGGCTACATAACCTGCCAACACTTAG

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

ATGCTAGATTATAATTATTTGTCTAAGGTGGAGATTACCAGGGTAGACTCGCTCTGGCCGTATGTAATGATGTCTGTGGCTACATAACCTGCCAACACTTAG

LINK-A PIP BM3

-/-

clone #6

LINK-A PIP BM3

+/+

PIP binding motif (nt. 1081-1140)3

--

38 nucleotides deletiond

Supplementary Figure 7 Generation of LINK-A PIP3-binding motif knockout cell line by CRISPR/Cas9 gene editing. (a) gRNA sequences targeting LINK-A PIP3-binding motif. (b) PCR analysis of targeted locus showing the band corresponding to the genomic deletion in colonies #1, 3 and

6. (c and d) Genotyping PCR results showing the deletion of LINK-A PIP3 binding region in colonies #3 (c) and #6 (d) of MDA-MB-231 cells. Unprocessed original scans of all gels with size marker are shown in Supplementary Fig. 9.





100

50

0

Su

rviv

al R

ate

(%

)

month

High LINK-A

Low LINK-A

p=0.0116

0 100 20050 150

0

20

40

60

80

100

120

LIN

K-A

Po

sitiv

e (

%)

NBT

Maligan

t

p=4.8e-4e f

g High Low

p-A

KT

(Th

r30

8)

p-A

KT

(Se

r47

3)

LIN

K-A

SNPs within gene loci significantly associated with survival of breast cancer in TCGA samples (n=712)LINK-A

chr1:238628248 rs12095274 G A 2.14(1.21-3.78) 0.008673 2.15E-08 765 137 16 5

ChrPosID SNPEffect

Allele

Reference

AlleleHR MissingeQTL-PP AA AG GG

SNPs within gene loci significiantly associated with survival of lung cancer in TCGA samples (LUAD+LUSC)LINK-A

(n=793)

chr1:238655389 rs2392862 T G 1.25(1.01-1.54) 0.042 0.744 433 299 61 0

eQTL-PEffect

Allele

Reference

AlleleHR PChrPosID SNP MissingGG GT TT

SNPs within gene loci significantly associated with lung cancerLINK-A

(n=2332) compared to normal lung tissues (n=3077)

chr1:238667409 rs7513497 A G 2.05(1.24-3.40) 0.0054

chr1:238666721 rs116316949 T C 3.21(1.30-7.91) 0.0113

chr1:238666024 rs10925671 C G 0.31(0.13-0.77) 0.0114

chr1:238665702 rs10925670 T C 3.21(1.30-7.93) 0.0116

chr1:238664537 rs10925669 A G 0.31(0.13-0.77) 0.0118

chr1:238667405 - T G 0.39(0.16-0.97) 0.0417

chr1:238667427 - C G 0.28(0.08-0.96) 0.0431

PChrPosID SNPEffect

Allele

Reference

AlleleOR

SNPs within gene loci significantly associated with gastric cancerLINK-A

(n=1006) compared to normal gastric tissues (n=2273)

chr1:238668602 rs11809478 T C 1.24(1.06-1.46) 0.008

chr1:238664492 rs59083989 G A 1.23(1.05-1.45) 0.011

OR PChrPosID SNPEffect

Allele

Reference

Allele

a

b

c

d

i

D7

**

D0 D1 D3 D5

0

1

2

3

4

Ab

so

rba

nce

(5

90

nm

)

�

CTL shRNA

LINK-A sh#2

LINK-A sh#3

MDA-MB-231

h

j

0

5

10

15

20

% o

fA

nn

ex-V

Po

sitiv

e **

CT

L

LIN

K-AsiRNA

Annex-V Positive

0

50

100

150

200

10 10 10 10 10 10101 2 3 4 5 76

FL1-A

Co

un

t

CTL si LINK-A si

k

Re

lative

Exp

ressio

n L

eve

l

CTL

LIN

K-A

MDA-MB-231

0

0.2

0.4

0.6

0.8

1.0

1.2 ***

siRNA

Re

lative

Exp

ressio

n L

eve

l

MDA-MB-468

0

0.2

0.4

0.6

0.8

1.0

1.2 ***

CTL

LIN

K-A

Re

lative

Exp

ressio

n L

eve

l

0

0.2

0.4

0.6

0.8

1.0

1.2

MDA-MB-231

*

CTL

LIN

K-A

#1shRNA

LIN

K-A

#2

LINK-A

Ne

ga

tive

Po

sitiv

e

Supplementary Figure 8 Identification of LINK-A SNP and its correlation with LINK-A expression level and phospho-AKT status in breast cancer tissues. (a) SNPs within LINK-A gene locus significantly associated with survival of breast cancer in TCGA samples (n=712 breast tumors; **p<0.01, two-tailed Wilcoxon test). (b) SNPs within LINK-A gene locus significantly associated with survival of lung cancer in TCGA samples (LUAD+LUSC) (n=793 lung tumors; *p<0.05, two-tailed Wilcoxon test). (c) SNPs within LINK-A gene locus significantly associated with lung cancer compared to normal lung tissues (n=2,332 lung tumors vs. n=3,077 normal lung tissues; *p<0.05 and **p<0.01, two-tailed Wilcoxon test). (d) SNPs within LINK-A gene locus significantly associated with gastric cancer () compared to normal gastric tissues (n=1,006 gastric tumors vs. n=2,273 normal gastric tissues; *p<0.05 and **p<0.01, two-tailed Wilcoxon test). (e) Breast cancer tissue microarrays were subjected to RNAscope® to detect LINK-A expression. Left panel: representative images. Scale bars, 200 µm. Right

panel: statistical analysis (n=3 independent tissue microarrays with 3, 20, 6 normal breast tissues and 37, 20, 35 breast tumors). (f) Kaplan-Meier survival analysis of LINK-A low and high breast cancer patients (n=84 and 66 patients respectively, log rank test). (g) Immunohistochemical staining of indicated phospho-AKT in breast cancer tissues. Scale bars, 200 µm. (h) RT-qPCR analyses of LINK-A expression level in MDA-MB-231 (left panel) and MDA-MB-468 (right panel) cells transfected with control or LINK-A siRNA. (i) RT-qPCR analyses of LINK-A expression level in MDA-MB-231 cells harboring control or LINK-A shRNA. (j) Cell proliferation rate was assessed by OD density (590 nm) in MDA-MB-231 cells harboring control or LINK-A shRNA. (k) Cell apoptosis rate was assessed by FACS in MDA-MB-231 cells transfected with control or LINK-A siRNA. For e, h, i, j and k, mean ± s.e.m. were derived from n=3 independent experiments (*p<0.05, **p<0.01, and ***p<0.001, two-tailed paired Student’s t-test). Statistics source data for e, f and k are in Supplementary Table 6.




Figure 2c

Cell Permeable IPn D

MS

OD

MS

OIn

s(1

,4,5

,6)P

4

Ins(

1,

,4,5

,6)P

53

DM

SO

DM

SO

Ins(

1,4

,5,6

)P4

Ins(

1,

,4,5

,6)P

53

DM

SO

DM

SO

Ins(

1,4

,5,6

)P4

Ins(

1,

,4,5

,6)P

53

GST-MS2BP - + - + - +

IB:GST-tag

Figure 2f

GST-MS2BP - +

WT

- + - + - + -

MS2-LINK-A DP

IP3

WT

DP

IP3

IB:GST-tag

+ - +

WT

DP

IP3

Figure 3f

siRNA CTL LINK-A

EGF 0 15 30 60 min0 15 30 60

IB: p-AKT(Thr308)

IB: p-AKT(Ser473)

IB: AKT

IB: Vinculin

IB: p-GSK-3b

-siRNA CTL LINK-A

EGF + - +

IB: p-AKT(Thr308)

IB: p-AKT(Ser473)

IB: AKT

Control PIP IB:AKT

PIIB:AKT

PI(3)PIB:AKT

PI(4)PIB:AKT

PI(5)PIB:AKT

PI(3,4)P2

IB:AKT

PI(3,5)P2

IB:AKT

PI(4,5)P2

IB:AKT

PI(3,4,5)P3

IB:AKT

-siRNA CTL LINK-A

EGF + - +

Figure 3h

IB: p-AKT(Thr308)

IB: p-AKT(Ser473)

IB: AKT

IB: p-PDK1(Ser241)

IB: p-PTEN(Ser308)

IB: p-c-Raf(Ser259)

IB: Vinculin

shRNA CTL

#2

#3

LINK-A

Figure 3g


Supplementary Figure 9 Unprocessed original scans of all blots




Figure 4g

IB:AKT

Strep.-HRP

Inp

ut

Be

ad

s

383G10

WT

D2

-5

3C

4A

6A

LINK-A

Figure 4i

IP:GST-tagIB:GST

IP:IgGIB:GST

IP:GST-tagIB:AKT

InputIB:AKT

- +WT

EGFMS2 DPIP3 3C 4A 6A

- + - + - + - + - +

Figure 5b

EGF - + - +PIK3CA +/+ +/-

IB:p-AKT(Thr308)

IB:p-AKT(Ser473)

IB:AKT

IB:PDK1

IB:GAPDHFigure 5e

EGF - + - + - + - +M

ock

WT

DPC

DPIP

3

IB:p-AKT(Thr308)

IB:p-AKT(Ser473)

IB:AKT

IB:PDK1

IB:GAPDH

LINK-A

Figure 5h

EGF - + - + - + - +

LINK-A PIP3 - - + + - - + +

- - - - + + + +

IB:p-AKT(Thr308)

IB:p-AKT(Ser473)

IB:AKT

IB:PDK1

IB:GAPDH

Figure 5k

LINK-AEGF - + - + - + - +

Moc

k

WT

DPC

DPIP

3

IB:p-AKT(Thr308)

IB:p-AKT(Ser473)

IB:AKT

IB:PDK1

IB:GAPDH

Figure 5l

EGF - + - + - + - +

RP11

-383G10

WT

- +

RNA 3C 4A 6A

LINK-A

PIP3 ++ ++ ++ ++ ++

IB:p-AKT(Thr308)

IB:p-AKT(Ser473)

IB:AKT

IB:PDK1

Figure 5m

IP:AKTIB:AKT

IP:IgGIB:AKT

InputIB:AKT

EGF - + - + - + - +

RP11

-383G10

WT

- +

3C 4A 6A

LINK-A

Supplementary Figure 9 Continued




Figure 6e

EGF - + - + - + - + - + - + - + - + - +

IB:AKT

LINK-A PIP -BM3 +/+ -/-,#3 -/-,#6 +/+ -/-,#3 -/-,#6 +/+ -/-,#3 -/-,#6

Input IP:AKT IP:IgG

Figure 6g

EGF - + - + - +

LINK-A PIP -BM3

+/+ -/-,#3 -/-,#6

IB:p-AKT(Ser473)

IB:p-AKT(Thr308)

IB:AKT

IB:PDK1

IB:GAPDH

Figure 7d

Perifosine(25 nM) - + - + - + - + - + - + - + - + - +

+/+ -/-,#3 -/-,#6 +/+ -/-,#3 -/-,#6 +/+ -/-,#3 -/-,#6

Input IP:AKT IP:IgG

IB:AKT

Supplementary Figure 2f

Permeable IPn DM

SO

DM

SO

Ins(

1,4

,5,6

)P4

Ins(

1,

,4,5

,6)P

53

DM

SO

DM

SO

Ins(

1,4

,5,6

)P4

Ins(

1,

,4,5

,6)P

53

DM

SO

DM

SO

Ins(

1,4

,5,6

)P4

Ins(

1,

,4,5

,6)P

53

GST-MS2BP - + - + - +

IB:PI3K p110a

Supplementary Figure 2i

GST-MS2BP - +F

L- + - + - + - + - +

MS2-LINK-A DP

IP3

FL

DP

IP3

FL

DP

IP3

IB:PI3K p110a

Supplementary Figure 3c

LY294002 - + - +

MD

A-M

B-

23

1

MD

A-M

B-

46

8

IB:AKT

IB:p-AKT(Ser473)

Mem

.

Cyto.

IB: Na/K ATPase

IB: a-Tubulin

SupplementaryFigure 4a

0.5

1

1.52

2.534569

(kb)

SupplementaryFigure 5d

Ma

rke

r

WT 3C 4A 6A

Biotinylated LINK-A





IB:p-GSK-3b(Ser9)

Blank Vector

LINK-A FL

LINK-A�DPIP3

EGF

IB:AKT

IB:p-AKT(Ser473)

IB:p-AKT(Thr308)

IB:GSK-3b

0 15 30 60 min0 15 30 60 0 15 30 60

SupplementaryFigure 6f

100

200300400500

1000

(bp

)

Ma

rke

r

1 2 3 4 5 6 7 WT

WT

-/-LINK-A PIP BM3

SupplementaryFigure 7b

SupplementaryFigure 6c

Bla

nk

Ve

cto

r

Bla

nk

Ve

cto

r

LIN

K-A

F

L

LIN

K-A

DP

IP3

EGF - + - + - + - +

IB:AKT

IB:p-AKT(Ser473)

IB:p-AKT(Thr308)

IB:p-GSK-3b(Ser9)

IB:GSK-3b





Supplementary Table Legends

Supplementary Table 1 Lipid-bound lncRNA profiling of breast cancers vs. normal adjacent tissues by LncRNA Array 3.0. Raw signal intensities were normalized in quantile method by GeneSpring GX v12.0, and low intensity LncRNAs were filtered (LncRNAs that at least 2 out of 2 samples have flags in Present or Marginal were chosen for further analysis). ProbeName: Probe Name. [1099714F] ~ [1099715F] (raw): Raw intensities of each sample. [1099714F] ~ [1099715F] (normalized): Normalized intensities of each sample (log2 transformed). GeneSymbol: LncRNA symbol name.

Supplementary Table 2 Changes in the LiP pattern of AKT PH domain upon PIP3, LINK-A or PIP3/LINK-A binding detected by LC-MS.Complete mass spectrometry analysis results for LiP assay with recombinant AKT PH domain (a.a. 1-111) in the presence of PIP3 alone or in combination with synthesized with wild-type LINK-A RNA oligonucleotide (nt.1,100-1,117) or indicated mutant. Peptide spectrum match numbers (#PSMs) from each experimental condition were shown.

Supplementary Table 3 Clinipathological parameters of tissue microarrays used in this study.HBre-Duc150Sur-01: Breast carcinoma, 150 cases, 1core/case, clinical stage I, II, III. Survival data followed up 9-12 years (US Biomax).T6235086-5: 37 different breast tumors and 3 corresponding normal control (Biochain);TMA007: Triple negative breast cancer tissue array, including TNM, clinical stage and pathology grade, majority cases with negative of ER, PR, HER-2 in IHC (Indivumed); Nanjing cohort: Breast cancer tissue array containing 20 breast cancer tissues (3 are triple negative), including TNM and clinical stage information, 20 cases/20 cores (Yixing people’s hospital); Duke cohort: 151 Frozen breast tumors samples with recurrence information, obtained 123 RNA isolation samples in all for qPCR assay, and all the breast cancer tissues were performed for tissue array by the core facility of MD Anderson, and 97 tissue samples were available in final for further assays (Duke University).

Supplementary Table 4: List of antibodies used in this study. Commercially available antibodies and their catalogue number, clone number and usage are listed.

Supplementary Table 5: List oligonucleotides used in this study. The sequence for all siRNAs/shRNAs/LNAs, primers for PCR/RT-qPCR/RIP/SNP detection, synthesized LINK-A RNA oligonucleotides are provided in this table.

Supplementary Table 6 Statistics source data. Raw data of the statistical analysis for experiments with representative images are provided.

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