NPV-BEZ235 PKI-587

GDC-0980 PD0325901

Supplemental Figure S1

Structures of Inhibitors NVP-BEZ235; PKI-587; GDC-0980 and PD0325901

pAKT Ser473

S6

-

ERK

AKT

- 0 .005 .01 .05 .1 .5 1

pAKT Thr308

Neurotensin + Insulin

pS6 Ser240/244

pERKT202/Y204

NPV-BEZ235 μM

Neurotensin + Insulin-

pAKT Ser473

S6

ERK

AKT

- 0 .005 .01 .05 .1 .5 1

pAKT Thr308

pS6 Ser240/244

pERKT202/Y204

Supplemental Figure S2

BA

NPV-BEZ235 μM

NPV-BEZ235 induces over-activation of ERK phosphorylation in MiaPaCa-2 (A) and PANC-1 (B) cells

NPV-BEZ235, µM - - 0.01 0.1 1 - - 0.01 0.1 1

PD 0325901 - - - - - + + + + +

Neurotensin+ Insulin

ERK

pERKT202/Y204

NPV-BEZ235

PD0325901, µM

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

Neurotensin+ Insulin

Supplemental Figure S3

ERK

pERKT202/Y204

- - .005 .01 .05 .1 - - .005 .01 .05 0.1

A

B

The MEK inhibitor PD 0325901 suppresses ERK over-activation induced by NPV-BEZ235 at nanomolar concentrations.

Supplemental Figure S4

BA

pMEKSer217/221

MEK

Neurotensin+ Insulin Neurotensin+ Insulin

1 - - .01 .1 1 1 - - .01 .1 1

- 1 5

NPV-BEZ 235, M

PD , M1 - 1 5 1

C D

pMEKSer217/221

MEK

Serum Serum

1 - - .01 .1 1 1 - - .01 .1 1

- 1 5

NPV-BEZ 235, M

PD , M1 - 1 5 1

Overactivation of MEK phosphorylation by combination of dual PI3K/mTOR kinase inhibitor NPV-BEZ235 and MEK inhibitor PD0325901.

B

Supplemental Figure S5

PKI GDC--

Neurotensin + Insulin

0 15 30 60

-

BEZ

Time, min

Neurotensin + InsulinA

Dual PI3K/mTOR kinase inhibitors NVP-BEZ235, PKI-587 and GDC-0980 suppress PIP3 accumulation in PDAC cells in different times of stimulation.

IGF

- +

OSI-906

EGF

Lapatinib

- +

EGF

AG-1478

- +

0

1

2

3

4

0.0

0.4

0.8

1.2

1.6

0.0

0.5

1.0

1.5

2.0

AG - + Lp - + OSI - +

Fo

ld i

nc

rea

se b

y B

EZ

Fo

ld i

nc

rea

se b

y B

EZ

A

DB C

Fo

ld i

nc

rea

se b

y B

EZ

Supplemental Figure S6

NPV-BEZ235 enhances ERK pathway activation through a pathway that does not require EGFR, HER2 or insulin/IGF-1 receptor