daniel et al suppl . fig . 1
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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+b In stimulated SMCs, STAT3 expression was found to be up-regulated 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).
STAT3
Tubulin
FCS - 8h 12h
a
0
2
4
6S
TAT3
m
RN
A ex
pres
sion
FCS - 4h 8h
*
*
b
mR
NA
exp
ress
ion
fold
cha
nge
control injured0
5
10160
165
170cyclin D1
mR
NA
exp
ress
ion
fold
cha
nge
Control Injured0
5
10100
105
110survivin
Daniel et al Suppl. Fig. 3
a*
*
b
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
- - - + + +- 2.5µM 5µM MOCK 2.5µM 5µM
0.00
0.05
0.10
0.15
0.20
0.25
0.30
SM
C a
popt
osis
(OD
405
nm)
FCSWP1066 (µM)
b
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 non-necrotic cells was determined by trypan blue exclusion (*P<0.05, n=4).
- + + + + +- MOCK 10 20 50 100
0.00
0.25
0.50
0.75
1.00
Frac
tion
of n
on-n
ecro
tic c
ells
FCSWP1066 (µM)
* *
*
*
*
Daniel et al Suppl. Fig. 5
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.).
Control WP10660
1
2
3
4
5
6
a
b
Inde
x of
re-e
ndot
helia
lizat
ion
(n=0
-6)
Daniel et al Suppl. Fig. 6
a
b
uninjured control uninjured WP1066
Uninjured WP1066 vWF uninjured WP1066 TUNEL50µm 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).
c
Daniel et al Suppl. Fig. 7
Control WP10663.00
4.00
Leukocytes109/L
Control WP10660.001.002.003.004.005.006.007.008.009.00
10.00
Erythrocytes1012/L
Control WP10660.00
50.00
100.00
150.00
Hemoglobing/L
Control WP10660.00
0.10
0.20
0.30
0.40
0.50
HematocritL/L
Control WP10660.00
10.00
20.00
30.00
40.00
50.00
60.00
MCVfL
Control WP10660.002.004.006.008.00
10.0012.0014.0016.0018.00
MCHpg
Control WP10660.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
MCHCg/dL
Control WP10660.00
100.00
200.00
300.00
400.00
500.00
600.00
Platelets109/L
a b
c d
e f
g h
Control WP10660.00
0.03
0.05
Eosinophils109/L
Control WP10660.000.100.200.300.400.500.600.700.80
Neutrophils109/L
Control WP10660.00
1.00
2.00
3.00
4.00
Lymphocytes109/L
Control WP10660.000.010.020.030.040.050.060.070.08
Monocytes109/L
Control WP10660.00
0.03
0.05
0.08
0.10
0.13
Creatininemg/dL
Control WP10660.00
5.00
10.00
15.00
20.00
25.00
Albuming/L
Control WP10660.00
100.00
200.00
300.00
400.00
500.00
600.00
LDHg/L
Control WP10660.00
40.00
80.00
120.00
160.00
200.00
ASTU/L
i j
k l
m n
o p
Control WP10660.00
5.00
10.00
15.00
20.00
25.00
30.00
ALTU/L
q
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.
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