31

Supplemental Figure Legends: Supplemental Figure 1. Caerulein treatment had similar acute effects. Caerulein (50 µg/kg) was

injected intraperitoneally at the time points shown (a). Histologic examination showed that within

24 hours after caerulein treatments, acute pancreatitis as indicated by edema and acinar cell

vacuolization developed in both control (b) and Acinar-Ras mice (c, 100X, inset 400X).

Measurements of pancreatitis parameters such as edema (d) and serum amylase (e) also indicated a

similar extent of acute inflammation after initial caerulein treatments.

Supplemental Figure 2. Inflammatory cell infiltration and fibrosis were evident in caerulein-

induced chronic pancreatitis in Acinar-Ras mice. 4 weeks after caerulein treatment pancreata of

Acinar-Ras mice showed strong infiltration of leukocytes (a, 100X, inset 400X), mainly

macrophages (b, 100X, inset 400X). Stroma replacement with positive collagen trichrome staining

(c, 100X) and abundant smooth muscle actin expression (d, 100X, inset 400X) was observed in

these animals. Total Ras was increased in acinar-Ras mice as compared with controls 4 weeks after

caerulein treatment (e).

Supplemental Figure 3. Caerulein treatments resulted in prolonged elevation of Ras downstream

signaling in the presence of mutant K-Ras. Phospho-Erk immunostaining indicated that increased

Erk activity was localized in acinar derived cells of pancreata from Acinar-Ras mice (a) compared

with control animals (b) at week 4 (100X, inset 400X). Caerulein caused prolonged elevation of

phosphor-Erk in Acinar-Ras mice as detected by western blot (c).

Supplemental Figure 4. LPS treatments led to chronic inflammation and precancerous lesions in

Acinar-Ras but not control mice. LPS (10 mg/kg) was injected intraperitoneally once per week for 4

consecutive weeks (a). Pancreata of control mice were histologically normal after LPS treatments

(b, 100X). In contrast, pancreata of Acinar-Ras mice developed chronic pancreatitis and PanINs

  32

within 4 weeks (c, 100X). Ras activity was strongly increased in pancreata of Acinar-Ras mice

after 4 weeks of LPS treatments (d) (*p<0.05 versus non-treated Acinar-Ras mice).

Supplemental Figure 5. NF-B inhibitory subunit IB- was phosphorylated after caerulein

induction in Acinar-Ras mice. After 2 series of caerulein treatments (week 4) increased

phosphorylation of IB- was detected by phosphor-specific antibody against IB- in pancreata of

Acinar-Ras mice (a, 200X) but not in controls (b, 200X).

Supplemental Figure 6. Cox-2 expression in pancreatic acinar cells led to chronic pancreatitis and

cancer in Acinar-Ras but not control mice. Pancreata of mice expressing Cox-2 alone were 2.5

times bigger than those of control littermates (a) (*p<0.05 compared to control mice, n=7 animals).

Cox-2 alone induced development of acinar cell vacuolization and cyst formation which was

observed after 8-months (b, 100X, inset 400X).

Supplemental Figure 7. Levels of active phospho-Erk increased in pancreata expressing both Cox-

2 and mutant K-Ras. Phospho-Erk was examined by immunohistochemical staining in the pancreata

expressing Cox-2. Cox-2 expression alone increased the level of phospho-Erk in many acinar cells

(a, 100X). The expression of oncogenic K-Ras alone increased phospo-Erk levels only in the rare

PanINs (b, 100X). The combination of expression of both Cox-2 and mutant K-Ras generated

much higher levels of phospho-Erk in acinar derived cells of 2-month-old mice (c,100X).

Supplemental Figure 8. Ras, Cox-2 and NF-B pathways examination in normal human pancreatic

tissues. p-Erk (a), Cox-2 (b), p65 translocation(c) and p-IB-(d) staining were not evident in

normal pancreas as compared with the strong staining of these signaling molecules in human

pancreatic cancer tissues shown in Figure 8.

Supplemental Figure 1

CaeruleinInjections Analysis5x 1x 1x 1x 1x

Days

5x 1x 1x 1x 1x

1 2 3 4 5 14 15 16 17 18 28a

b c

WTCer 24h

Acinar-RasCer 24h

1.5

ControlAcinar-Ras

gh

t)

d

150

200

ControlAcinar-Ras

(%)

e

0.5

1.0

Pan

crea

s(%

of

Bo

dy

Wei

g

50

100

150

Am

yla

se

ac

tivit

y

0.0Cearulein - + - +

0Cearulein - + - +

A

b

Supplemental Figure 2

a

c d

Acinar-Ras 4 weeks post CerLeukocytes

Acinar-Ras 4 weeks post CerMacrophages

c d

Acinar-Ras 4 weeks post CerCollagen

Acinar-Ras 4 weeks post CerSmooth muscle actin

e

Acinar-RasControl

Collagen Smooth muscle actin

Total Ras

GAPDH

Supplemental Figure 3

ba

Control Acinar-Rasc

Acinar-Ras 4 weeks post Cerp-Erk (Brown)

WT 4 weeks post Cerp-Erk

400 C t l

p-ERK

t-ERK

0 2 4 0 2 4 Time (Week)

*

200

300

400 ControlAcinar-Ras

o-E

RK

/To

tal-

ER

K%

of

co

ntr

ol

*

WEEK 0 WEEK 2 WEEK 40

100

Ph

os

ph %

Supplemental Figure 4

LPS

Days 1 14 28217a

LPSinjections Analysis1x 1x 1x 1x

b cb c

WT 4 weeks post LPS Acinar-Ras 4 weeks post LPS

- - - + + + Ras-GTP

GAPDH

LPSd

400

600

Ras

/ G

AP

DH

of c

ontr

ol

*

0

200

LPS - +

Act

ive

R%

o

Supplemental Figure 5

ba

Acinar-Ras 4 weeks post Cerp-IKB- (Brown)

WT 4 weeks post Cerp-IKB-

Supplemental Figure 6

2.5

a

*

1.0

1.5

2.0

Pa

nc

rea

s%

of

Bo

dy

We

igh

t)

Control0.0

0.5(%

b

Cox-2Tg

Cox-2Tg

Supplemental Figure 7

ba

p-Erk p-Erk

Cox-2Tg Acinar-Ras

c

A i R C 2

p-Erk

Acinar-Ras-Cox-2

Supplemental Figure 8

a b

c d

Normal human pancreasIHC: p-Erk

Normal human pancreasIHC: Cox-2

Normal human pancreasIHC: p-IKB-

Normal human pancreasIHC: p65