Daniel et al Suppl. Fig. 1
a
b
a Co-staining for p-STAT3 and α-smooth muscle actin
revealed that p-STAT3 is predominantly expressed in
neointimal SMCs. b Staining of STAT3 was also detected in
regions of high macrophage density, particularly within the
medial layer. The overlay image comprises an overlay of
STAT3/DAPI together with a CD68 staining of a subsequent
slide.
Daniel et al Suppl. Fig. 2
a
*
STAT3
mRNA expression
6
b
*
4
STAT3
2
Tubulin
0
FCS
-
4h
8h
FCS
-
8h
12h
a+b In stimulated SMCs, STAT3 expression was found to be upregulated on the mRNA level at 4 and 8 hours and on the protein level
at 8 and 12 hours after stimulation using real-time PCR and Western
blotting , respectively (*P<0.05, n=4).
Daniel et al Suppl. Fig. 3
b
cyclin D1
170
*
165
160
10
5
0
control
injured
mRNA expression fold change
mRNA expression fold change
a
survivin
110
*
105
100
10
5
0
Control
Injured
a+b Real-time PCR showed a significant up-regulation of cyclin D1 and
survivn mRNA levels in the dilated artery at 3 weeks after dilation
(*P<0.05, n=4).
Daniel et al Suppl. Fig. 4
a
0.35
*
SMC apoptosis
(OD 405nm)
0.30
*
0.25
0.20
0.15
0.10
0.05
0.00
FCS
WP1066 (µM)
b
-
2.5µM
5µM
+
MOCK
+
2.5µM
*
*
+
5µM
Fraction of non-necrotic cells
1.00
0.75
0.50
0.25
0.00
FCS
WP1066 (µM)
*
-
+
MOCK
+
10
+
20
+
50
+
100
a SMCs were incubated in basal medium or growth medium
supplemented with FCS in the absence or presence of different
concentrations of WP1066 for 24 h. Apoptosis of SMCs was evaluated
by a TUNEL-based cell death detection ELISA (*P<0.05, n=4). b
SMCs were incubated with growth medium in the absence or
presence of different concentrations of WP1066, and the fraction nonnecrotic cells was determined by trypan blue exclusion (*P<0.05, n=4).
Daniel et al Suppl. Fig. 5
a
Index of re-endothelialization
(n=0-6)
b
6
5
4
3
2
1
0
Control
WP1066
a Representative cross sections of femoral arteries from control
mice (left) or mice treated with WP1066 (right) stained for CD31
(PECAM-1) at 3 weeks after dilation. b Re-endothelialization
was determined by estimating the lumen coverage on a scale of
0-6 (0, no coverage; 6, complete coverage) (n=6, P=n.s.).
Daniel et al Suppl. Fig. 6
a
uninjured control
uninjured WP1066
c
b
Uninjured WP1066 vWF
50µm
uninjured WP1066 TUNEL
50µm
a Representative en face staining with Evans blue of uninjured femoral
arteries from controls (left) or after local application of WP1066 (right).
b The integrity of the endothelial layer after application of WP1066 was
also confirmed by staining for endothelial markers (arrowhead indicates
staining for von-Willebrand-factor). c Apoptotic cell death was very rare
after application of WP1066 to uninjured arteries and occurred mainly
in the adventitia and neighboring tissue but not within the intima or
media (arrow indicates TUNEL-positive cell in the neighboring tissue).
Daniel et al Suppl. Fig. 7
a
109/L
b
Leukocytes
Erythrocytes
5.00
1012/L 12.00
4.00
10.00
8.00
3.00
6.00
2.00
4.00
1.00
2.00
0.00
0.00
Control
c
g/L
WP1066
Control
d
Hemoglobin
WP1066
Hematocrit
L/L 0.50
150.00
0.40
100.00
0.30
0.20
50.00
0.10
0.00
0.00
Control
e
fL
WP1066
Control
f
MCV
60.00
pg
50.00
40.00
30.00
20.00
10.00
0.00
Control
g
g/dL
MCH
18.00
16.00
14.00
12.00
10.00
8.00
6.00
4.00
2.00
0.00
WP1066
Control
h
MCHC
35.00
109/L
WP1066
Platelets
700.00
30.00
600.00
25.00
500.00
20.00
400.00
15.00
300.00
10.00
200.00
5.00
100.00
0.00
WP1066
0.00
Control
WP1066
Control
WP1066
i
j
Eosinophils
Neutrophils
109/L 1.00
109/L 0.10
0.80
0.08
0.60
0.05
0.40
0.03
0.20
0.00
0.00
Control
Lymphocytes
k
Control
WP1066
Monocytes
l
109/L 4.00
WP1066
109/L 0.25
0.20
3.00
0.15
2.00
0.10
1.00
0.05
0.00
0.00
Control
m
WP1066
Control
n
Creatinine
Albumin
mg/dL 0.15
g/L 30.00
0.13
25.00
0.10
20.00
0.08
15.00
0.05
10.00
0.03
5.00
0.00
0.00
Control
o
WP1066
Control
WP1066
p
LDH
g/L 700.00
WP1066
AST
U/L 250.00
600.00
200.00
500.00
400.00
150.00
300.00
100.00
200.00
50.00
100.00
0.00
0.00
Control
WP1066
Control
WP1066
q
ALT
U/L 30.00
25.00
20.00
15.00
10.00
5.00
0.00
Control
WP1066
a-q Blood tests were performed at 1 week after injury, in order to
analyze the systemic effects of WP1066. There was no significant
difference between vehicle (DMSO) and WP1066 treated mice.