IP: IgG AEG-1

IP

Input

AEG-1

Akt3

AEG-1

Akt3

IB:

Supplementary Figure 1

A

B

Supplementary Figure 2

Supplementary Figure 3

Blind scores of TMA AEG-1 and Akt2

3 4 5 6 70

2

4

6

8

NB GI GII GIII GIV

r2=0.9221

AEG-1 scores

Akt

2 sc

ore

s

AEG-1

Myc tag

Akt2

Actin

p-Akt2-S474

Vect

or C

ontr

ol

Akt2

-PH

Akt1

-PH

AEG

-1

Akt2

-PH

+AEG

1

Akt1

-PH

+ AE

G1

Ctrl

siRN

A

Akt2

-siR

NA

Ctrl

-siR

NA

Akt1

-siR

NA

Akt2

p-Akt2-S474

Akt1

ttAkt

Actin

p-Akt-S473

A B

AEG-1

Akt2

Akt1

Actin

AEG-1 — — + — + +Akt2-siRNA — + + — — —Akt1-siRNA — — — + + —

AEG-1

Akt2

Akt1

Actin

Myc tag

AEG-1 — — + — + +Akt2-PH — + + — — —Akt1-PH — — — + + —

C D

Supplementary Figure 4

Supplementary Figure 5

0 1 2 30.0

0.5

1.0

1.5 Vector ControlAkt2-PH

Cell Proliferation Assay

Akt2-PH+AEG-1Akt1-PH

*

Akt1-PH+AEG-1AEG-1

Days

OD

490

nm

0 1 2 30

1

2

3

4 CtrlAkt2-siRNA

Cell Proliferation Assay

Akt2-siRNA+AEG-1Akt1-siRNAAkt1-siRNA+AEG-1AEG-1

Days

OD

490

nm

A

B

0’ 15’ 30’ 1h 2h 3h 0’ 15’ 30’ 1h 2h 3h 0’ 15’ 30’ 1h 2h 3h

p-Akt S473

p-Akt T308

ttAkt

Actin

Control AEG-1-siRNAAEG-1 OE

Ctrl AEG-1 OE AEG-1-siRNA

S473 1/slope -260.0 -321.2 -223.6

T308 1/slope -220.3 -224.0 -226.9

P values (S/T) 0.0038/0.0228 0.0003/0.0295 0.0424/0.0598

Linear regression analysis of p-Akt S473 and T308 half-life

Supplementary Figure 6

+LY294002:

A

B

Supplementary Figure 7

AEG-1 — — — — + + + +Akt2-PH — + — + — + — +CAPE — — + + — — + +

AEG-1

p-Akt2-S474

Akt2

p-P65

P65

Actin

Supplementary Figure 8

Supplementary Table 1

S-Table 2 Source: US BIOMAX GL801 Brain tumor TMA Scores of AEG1 and Akt staining

Position Gender Age Organ Pathology Grade Type AEG1 score Akt2 score

A1 F 68 Brain Astrocytoma of right temporal lobe II Malignant 4 3

A2 F 44 Brain Ganglioglioma of right frontal lobe - Malignant 5 4

A3 F 36 Brain Astrocytoma of left temporal lobe II Malignant 8 6

A4 M 41 Brain Astrocytoma of right frontal lobe I Malignant 3 3

A5 F 14 Brain Astrocytoma II Malignant 2 2

A6 M 39 Brain Astrocytoma I Malignant 4 5

A7 M 36 Brain Astrocytoma of frontal lobe I Malignant 3 2

A8 F 30 Brain Astrocytoma I Malignant 2 2

A9 F 37 Brain Astrocytoma of right temporal lobe II Malignant 2 2

A10 M 52 Brain Astrocytoma of brain I Malignant 3 3

B1 F 42 Brain Astrocytoma of left occipital parietal lobe II Malignant 4 5

B2 M 16 Brain Astrocytoma of right occipital lobe II Malignant 5 5

B3 M 47 Brain Ganglioglioma of left temporoparietal lobe - Malignant 7 6

B4 M 17 Brain Astrocytoma of fourth ventricle II Malignant 5 9

B5 M 31 Brain Astrocytoma of left temporal lobe II Malignant 2 2

B6 F 34 Brain Astrocytoma II Malignant 3 2

B7 F 47 Brain Astrocytoma of left parietal lobe III Malignant 6 6

B8 F 34 Brain Colloid cell hyperplasia of left brain - Malignant 4 3

B9 F 40 Brain Astrocytoma of right temporoparietal lobe III Malignant 3 3

B10 F 40 Brain Astrocytoma of frontal lobe III Malignant 2 3

C1 M 53 Brain Astrocytoma of foramen magnum III Malignant 5 4

C2 M 58 Brain Astrocytoma of left temporal lobe II Malignant 8 6

C3 M 33 Brain Astrocytoma II Malignant 9 7

C4 M 43 Brain Brain tissue - Malignant 5 5

C5 F 48 Brain Astrocytoma of right temporal lobe II Malignant 5 4

C6 F 30 Brain Astrocytoma of fourth ventricle II Malignant 3 4

C7 M 53 Brain Astrocytoma of right temporal lobe II Malignant 4 5

C8 M 65 Brain Astrocytoma III Malignant 4 4

C9 F 33 Brain Astrocytoma of left temporoparietal lobe II Malignant 5 5

C10 M 43 Brain Astrocytoma of right cerebral ventricle II Malignant 2 2

D1 M 40 Brain Oligodendroglioma of right frontal lobe - Malignant 6 4

D2 M 39 Brain Astrocytoma of brain III Malignant 6 4

D3 F 36 Brain Astrocytoma of brain III Malignant 8 10

D4 F 36 Brain Astrocytoma of left temporal lobe II Malignant 5 3

D5 M 53 Brain Astrocytoma of left temporal lobe II Malignant 6 3

D6 F 47 Brain Astrocytoma of intracranial II Malignant 4 3

D7 F 16 Brain Astrocytoma of cerebellum III Malignant 3 5

D8 M 39 Brain Astrocytoma of left temporal lobe III Malignant 3 3

D9 M 35 Brain Astrocytoma with necrosis of left frontal lobe III Malignant 4 3

Supplementary Table 2

D10 M 59 Brain Astrocytoma of left temporal lobe III Malignant 4 5

E1 M 48 Brain Astrocytoma of temporoparietal lobe III Malignant 9 7

E2 M 36 Brain Astrocytoma of right cerebelli III Malignant 9 9

E3 F 51 Brain Astrocytoma III Malignant 8 5

E4 M 47 Brain Astrocytoma III Malignant 10 6

E5 M 44 BrainBrain tissue with colloid cell hyperplasia of right temporal lobe - Malignant 8 7

E6 M 68 Brain Astrocytoma III Malignant 7 5

E7 F 41 Brain Astrocytoma of brain III Malignant 8 6

E8 M 60 Brain Astrocytoma of left frontal lobe III Malignant 5 5

E9 M 53 Brain Astrocytoma of brain II Malignant 4 4

E10 F 48 Brain Astrocytoma of left frontal lobe III Malignant 2 4

F1 M 35 Brain Astrocytoma of left temporal lobe II Malignant 5 2

F2 M 40 Brain Astrocytoma of left occipital III Malignant 8 5

F3 F 47 Brain Astrocytoma of left temporal lobe III Malignant 8 7

F4 M 9 Brain Astrocytoma of right temporal lobe IV Malignant 10 9

F5 F 48 Brain Astrocytoma of left temporal lobe III Malignant 9 7

F6 M 68 Brain Astrocytoma of right temporal lobe III Malignant 9 8

F7 F 49 Brain Astrocytoma of right temporal lobe III Malignant 9 9

F8 M 5 Brain Astrocytoma of fourth ventricle IV Malignant 9 6

F9 M 76 Brain Astrocytoma II Malignant 4 4

F10 F 30 Brain Astrocytoma of temporal lobe IV Malignant 5 3

G1 M 45 Brain Tumor necrosis tissue of right frontal region - Malignant 8 5

G2 M 53 Brain Astrocytoma of left occipital parietal lobe III Malignant 10 8

G3 M 33 Brain Malignant oligodendroglioma - Malignant 9 5

G4 M 17 Brain Desmoplastic astrocytoma of left temporoparietal lobe - Malignant 9 7

G5 M 44 Brain Astrocytoma of intracranial II Malignant 6 5

G6 F 39 Brain Ependymoblastoma of posterior fossa - Malignant 10 9

G7 M 2 Brain Ependymoma of brain parietooccipital - Malignant 7 7

G8 M 48 Brain Ependymoma of brain - Malignant 8 7

G9 M 72 Brain Ependymoma of right parietal lobe - Malignant 7 7

G10 F 13 Brain Ependymoma of intracranial - Malignant 6 7

H1 F 68 Brain Matched cerebrum tissue of No.01 - Normal 3

H2 F 44 Brain Matched cerebrum tissue of No.02 - Normal 3 3

H3 F 36 Brain Matched cerebrum tissue of No.03 - Normal 5 4

H4 M 41 Brain Matched cerebrum tissue of No.04 - Normal 5 3

H5 F 33 Brain Normal cerebrum tissue - Normal 4 3

H6 F 37 Brain Normal cerebrum tissue - Normal 4 5

H7 M 39 Brain Normal cerebrum tissue - Normal 2 2

H8 M 52 Brain Normal cerebrum tissue - Normal 4 3

H9 F 47 Brain Normal cerebrum tissue - Normal 4 4

H10 M 72 Brain Normal cerebellum tissue - Normal 3 3

Supplementary Figure 9

+LY294002: 0’ 15’ 30’ 1h 2h 3h

Control

AEG-1 OE

AEG-1 KD

p-Akt1 S473

Akt1

Actin

50 100 150 200

-0.5

0.0

0.5

1.0

1.5

CtrlAEG-1 OEAEG1 siRNA

1/slope=-197.1

1/slope=-192.6

1/slope=-196.1

Time (mintues)

Rel

ativ

e fo

ld c

han

ges