Cosen-Binker, L. et al. Supplemental Figure 1dm5migu4zj3pb.cloudfront.net/.../JCI0834672sd.pdf ·...
Transcript of Cosen-Binker, L. et al. Supplemental Figure 1dm5migu4zj3pb.cloudfront.net/.../JCI0834672sd.pdf ·...
Cosen-Binker, L. et al. Supplemental Figure 1
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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
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ten
sity
Munc18cSNAP-23
VAMP-8
VAMP-2
Syn-4
I.P. anti-Syntaxin-4 Total lysate
Rel
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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
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e In
ten
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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
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ten
sity
Syn-2
SNAP-23
Munc18bVAMP-8 VAMP-2
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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.