Cosen-Binker, L. et al. Supplemental Figure 1

00.20.40.60.8

1

Rel

ativ

e In

ten

sity

ActinVAMP-8 VAMP-2

**

VAMP-8 + / +

VAMP-8 - / -

Cosen-Binker, L. et al. Supplemental Figure 2

CD / Saline

ED / SalineCD / Atp+CchCD / Cch

ED / Atp+CchED / Cch

CD / Saline

ED / SalineCD / Atp+CchCD / Cch

ED / Atp+CchED / Cch

D

0

1

2

3

4

5

# ZG

on

LPM

/ c

ell s

ectio

n

> 0 > 2 > 4 > 6 > 8 > 10 > 12 > 14

Distance ( µm )

0

1

2

3

4

5

# ZG

on

LPM

/ c

ell s

ectio

n

> 0 > 2 > 4 > 6 > 8 > 10 > 12 > 14

Distance ( µm )

VAMP-8 + / + VAMP-8 - / -

C

# ZG

on

LPM

/ c

ell s

ectio

n

VAMP-8 + / + VAMP-8 - / -0

5

10

15

20

25 **

A#

ZG /

cell

sect

ion

VAMP-8 + / + VAMP-8 - / -0

50

100

150

200

250 **

*** *

B

0

5

10

15

20

25

> 0 > 2 > 4 > 6 > 8 > 10 > 12 > 14

# ZG

/ ce

ll se

ctio

n

Distance ( µm )

# ZG

/ ce

ll se

ctio

n

Distance ( µm )

0

10

20

30

40

> 0 > 2 > 4 > 6 > 8 > 10 > 12 > 14

VAMP-8 + / + VAMP-8 - / -

Cosen-Binker, L. et al. Supplemental Figure 2

CD / Saline

ED / SalineCD / Atp+CchCD / Cch

ED / Atp+CchED / Cch

E#

ZG -

ZG fu

sion

s /

cell

sect

ion

VAMP-8 + / + VAMP-8 - / -0

5

10

15**

F

VAMP-8 + / + VAMP-8 - / -

Api

cal L

umen

Are

a ( µ

m2

)

0

5

10

15

20 ** **

Cosen-Binker, L. et al. Supplemental Figure 3

VAMP-8 + / +

a: no 1º Abb: KRHc: EtOH+Cch

VAMP-8 - / -

d: no 1º Abe: KRHf: EtOH+Cch

0

0.2

0.4

0.6

0.8

1 **

**

**

** **

a b c d e f a b c d e f a b c d e f a b c d e f a b c d e f

Rel

ativ

e In

ten

sity

Munc18cSNAP-23

VAMP-8

VAMP-2

Syn-4

I.P. anti-Syntaxin-4 Total lysate

Rel

ativ

e In

ten

sity

0

0.2

0.4

0.6

0.8

1

a b c d e f a b c d e f a b c d e f a b c d e f a b c d e f

*

**

**

*

**

***

Munc18c

SNAP-23

VAMP-8

VAMP-2Syn-4

A

C

VAMP-8 + / +

a: no 1º Abb: KRHc: EtOHd: Cche: EtOH+Cch

Total lysate

0

0.2

0.4

0.6

0.8

1

a b c d e a b c d e a b c d e a b c d e a b c d e

Rel

ativ

e In

ten

sity

Syn-3

SNAP-23

Munc18bVAMP-8

VAMP-2

I.P. anti-Syntaxin-3

Rel

ativ

e In

ten

sity

0

0.2

0.4

0.6

0.8

1

a b c d e a b c d e a b c d e a b c d e a b c d e

**

**

**

**

**

Syn-3SNAP-23

Munc18b

VAMP-8

VAMP-2

B

VAMP-8 + / +

a: no 1º Abb: KRHc: EtOHd: Cche: EtOH+Cch

Total lysate

0

0.2

0.4

0.6

0.8

1

a b c d e a b c d e a b c d e a b c d e a b c d e

Rel

ativ

e In

ten

sity

Syn-2

SNAP-23

Munc18bVAMP-8 VAMP-2

00.20.40.60.8

11.21.4

a b c d e a b c d e a b c d e a b c d e a b c d e

Rel

ativ

e In

ten

sity

**

**

**

**

Syn-2

SNAP-23

Munc18b

VAMP-8

VAMP-2

I.P. anti-Syntaxin-2

Cosen-Binker, L. et al. Supplemental Table 1

Group

below below belowControl 0±0 0.16±0.07 detection detection detection 0.79±0.10

limit limit limit

Cerulein 21.05±0.84 5.48±0.29 439±25 6.13±0.10 357±33 25.21±0.23

below below belowControl 0±0 0.11±0.04 detection detection detection 0.75±0.08

limit limit limit

Cerulein 7.32±0.94 4.17±0.16 176±18 2.85±0.14 130±16 16.03±0.15

MPOUMPO/mg

DNA

Active Trypsin

nmoles/mgDNA

NF-kB (p65)

ug/mg DNA

NF-kB (p50)

ug/mg DNA

LPOnmol/mg

DNA

MDAumol/mg

DNA

VAMP-8+/+

VAMP-8-/-

∗ ∗ ∗ ∗ ∗

Supplemental Table 1. Pancreatic tissue parameters in supramaximal cerulein-induced pancreatitis

∗ p<0.05

Cosen-Binker, L. et al. Supplemental Table 2

Supplemental Table 2. Serum parameters insupramaximal cerulein-induced pancreatitis

∗ p<0.05

∗ ∗ ∗ ∗ ∗

below belowControl 583±11 21±3 detection detection 7±2

limit limit

Cerulein 5689±48 4115±29 1144±39 523±32 991±42

below belowControl 525±19 16±2 detection detection 5±0.7

limit limit

Cerulein 2864±33 1806±28 699±12 273±39 611±21

VAMP-8+/+

VAMP-8-/-

Group Amylase(IU/L)

Lipase(IU/L)

IL-6(pg/mL)

TNFα(pg/mL)

MIP-1 α(pg/mL)

Cosen-Binker, L. et al. Supplemental Table 3

Supplemental Table 3. Histopathological score in supramaximal cerulein-induced pancreatitis

∗ p<0.05

Control 0±0 0±0 0±0 0±0

Cerulein 1.17±0.09 3.11±0.15 2.51±0.12 3.31±0.2

Control 0±0 0±0 0±0 0±0

Cerulein 0.97±0.05 1.54±0.16 1.17±0.09 1.32±0.14

VAMP-8+/+

VAMP-8-/-

Group Edema Hemorrhage Infiltrate Necrosis

∗ ∗ ∗ ∗

Cosen-Binker, L. et al. Supplemental Table 4

Supplemental Table 4. Lung tissue parameters and histopathological scorein supramaximal cerulein-induced pancreatitis

∗ p<0.05

∗ ∗ ∗ ∗ ∗ ∗

below belowControl 0±0 0±0 0±0 0±0 detection detection

limit limit

Cerulein 2.2±0.3 3.7±0.6 2.1±0.8 13.0±0.4 315±46 3.3±0.50

below belowControl 0±0 0±0 0±0 0±0 detection detection

limit limit

Cerulein 0.8±0.1 1.6±0.5 1.1±0.3 5.6±1.0 108±12 1.5±0.14

VAMP-8+/+

VAMP-8-/-

Group Edema Hemorrhage LeukocyteInfiltrate

MPOUMPO/mg

DNA

NF-kB (p65)ug/mg DNA

NF-kB (p55)ug/mg DNA

Cosen-Binker LI, Binker MG, Wang CC, Hong W and Gaisano HY. 1

Supplementary Data

Supplemental Figure 1: Summary of the NIH-Image densitometry analysis of the data from

Figure 3A showing VAMP-8, VAMP-2 and actin levels in pancreatic acini of VAMP-8+/+ and

VAMP-8-/- mice. The data was from three independent experiments, with samples performed in

duplicates. *: p <0.05.

Supplemental Figure 2: Morphometric analysis of EM of pancreatic tissues of CD- and ED-

fed VAMP-8+/+ and VAMP-8-/- mice: ZG abundance, distribution, fusion events, and apical

lumen area. The data shown is the analysis of the EM images shown in Figs. 6 and 7.

A) Number of ZGs per EM cell section. B) Distribution of ZGs within the acinar cell section. X

axis indicates the distance from the apical lumen. C) Number of ZGs per cell section docked on

the lateral PM (LPM). D) Distribution of ZGs per cell section docked on the LPM. X axis

indicates the distance from the junctional complex. E) Number of ZG-ZG fusions per EM cell

section. F) Apical lumen areas. For each strain and treatment, 300 cells from 3 independent

experiments were quantified. Data are expressed as means ± SD; statistical significance was

examined by Mann-Whitney U-test (non-parametric) for ZGs analysis and Student t test for

apical lumen areas. *: p<0.05 and **: p<0.01 compared to CD/Saline.

Supplemental Figure 3: Summary of the NIH-Image densitometry analysis of the data from

Figure 9, which are immunoprecipitated Syntaxin-4 (in A) and Syntaxin-2 (in B) from PM

fractions, and immunoprecipitated Syntaxin-3 from ZG membrane fractions (in C) prepared from

pancreatic acini stimulated by the indicated treatment conditions. VAMP-8+/+ and VAMP-8-/-

Cosen-Binker LI, Binker MG, Wang CC, Hong W and Gaisano HY. 2

mice acini were used in A, whereas only VAMP-8+/+ mice acini were used in B and C. The data

came from blots of three independent experiments, with samples performed in duplicates. Data

is expressed as means ± SD; *: p <0.05 compared to KRH.

Supplemental Table 1: Pancreatic tissue parameters in supramaximal cerulein-induced

pancreatitis.

*: p<0.05 compared to VAMP-8+/+ mice.

Supplemental Table 2: Serum parameters in supramaximal cerulein-induced pancreatitis.

*: p<0.05 compared to VAMP-8+/+ mice.

Supplemental Table 3: Histopathological score in supramaximal cerulein-induced

pancreatitis.

*: p<0.05 compared to VAMP-8+/+ mice.

Supplemental Table 4: Lung tissue parameters and histopathological score in

supramaximal cerulein-induced pancreatitis.

*: p<0.05 compared to VAMP-8+/+ mice.