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SUPPLEMENTAL FIGURES:
S1a
0nM
2nM
5nM
10nM
20nM
50nM
S1b
SH-EP/Surv
SH-EP/Ctr
0
10
50
0
2
5
10
20
50
nM YM155
Survivin
100
25
14 1200 965 798 554 361 133
% of untreated Ctr
GAPDH
Supplemental Figure 1: Dose-dependent effects of YM155 on mitochondrial structure and
Survivin protein levels. SH-EP/SurvYFP cells were treated for 24 hours with increasing
concentrations of YM55 (2-50 nM). (a) Mitochondrial structures were analyzed by CMXRos
staining (300 nM) via live cell microscopy using an Axiovert200M microscope equipped
with an ApoTome.2 system (Zeiss, Vienna, Austria). (b) Cell lysates were analyzed for
Survivin expression by immunoblot, GAPDH served as loading control. Densitometric
analyses were performed in the LabWorks software (UVP, Cambridge, UK).
S1c
30
% apoptosis
25
24h
SH-EP/Ctr
SH-EP/Surv
20
15
10
*
5
*
0
untreated
5nM
25nM
50nM
YM155
S1d
60
% apoptosis
50
48h
SH-EP/Ctr
SH-EP/Surv
40
***
30
***
20
10
**
0
untreated
5nM
25nM
50nM
YM155
Supplemental Figure 1: Repression of Survivin by YM155 induces apoptosis. SHEP/Ctr or SH-EP/Surv cells were treated with increasing concentrations of the
Survivin-inhibitor YM155 for either 24 (c) or 48 (d) hours. The mean of five
independent PI-FACS experiments is shown. Statistical differences between SHEP/Ctr and SH-EP/Surv cells were assessed by students t-test (***P<0.001,
**P<0.01, *P<0.05).
cell number [% of untreated control]
S2
120
untreated
100
2DG
***
w/oG
80
60
ns
40
20
0
SHEP/Ctr
SHEP/Surv
Supplemental Figure 2: SH-EP/Ctr cells and SH-EP/Surv cells were treated either
with 15 mM 2DG or were cultivated in glucose-free media for 24 hours.
Afterwards cell number was assessed by Casy® Cell counter (double
measurements for each probe). Shown is the mean of four independent
experiments. Statistical differences between SH-EP/Ctr and SH-EP/Surv cells
were assessed by students t-test (***P<0.001).
S3
2DG
0
1
2
2DG+LY294002 LY294002
4
1
2
4
1
4
h
Survivin
100
55
31
17
89
54
22
105
80
in % of Ctr
GAPDH
Supplemental Figure 3: Survivin as target for autophagosomal degradation. SHEP/Surv cells were treated with 15 mM 2DG alone or in combination with 40 µM
LY204002 for 0/1/2/4 hours. After cell lyses, Survivin expression was analyzed by
immunoblot, GAPDH served as loading control.
cell number [% of unterated control]
S4
120
untreated
QVD
100
Necro
QVD+Necro
80
60
40
20
0
- 2DG
+ 2DG
cell number [% of unterated control]
SH-EP/Ctr
120
untreated
QVD
100
Necro
QVD+Necro
80
60
40
20
0
- 2DG
+ 2DG
SH-EP/Surv
Supplemental Figure 4: Cell number after 2DG treatment. SH-EP/Ctr (upper panel) and SHEP/Surv cells (lower panel) were treated for 24 hours with 15 mM 2DG with or without
pre-incubation of 10 µM QVD, 20 µM Necrostation or a combination of both. Cell number
was assessed by counting in a Casy® Cell counter System of four independent experiments.
S5
Supplemental Figure 5: Survivin is a candidate for lysosomal degradation due to
the basicity of its C-terminal coiled coil domain. Basic amino acids (Lys, Arg) in the
coiled coil domain of Survivin are highly conserved among different mammalian
species. Basic amino acids are shown in black (human), red (mouse) and green
(rat).
S6a
SHEP/Surv-shParkin
Ctr
cl13
0
4
0
4 h 2DG
Survivin
pParkin
GAPDH
S6b
SH-EP/Surv
cyto
-
+
SH-EP/Surv-shParkin-cl13
mito
-
cyto
+
-
mito
+
-
+
4h 15mM 2DG
Survivin
CoxIV
α-Tubulin
Supplemental Figure 6: Survivin-degradation is mediated by Parkin. SHEP/Surv-shCtr and SH-EP/Surv-shParkin-cl13 cells were treated with 15 mM
2DG for four hours. (a) Whole cell lysates were analyzed for Survivin and
pParkin expression. GAPDH served as loading control. (b) Mitochondrial and
cytoplasmic lysates were analyzed for Survivin. CoxIV (mito) and Tubulin
(cyto) served as purification controls.
S7a
60
untreated
5 nM YM155
10 nM YM155
20 nM YM155
50 nM YM155
100 nM YM155
% apoptosis
50
40
30
20
10
0
24h
48h
72h
S7b
0
4
8
16
24
h YM155 50 nM
Survivin
Tubulin
Supplemental Figure 7: Repression of endogenous Survivin by YM155
effectively induces cell death. (a) STA-NB15 cells were treated with
increasing concentrations of YM155 (5 -100 nM). Cell death was analyzed
by FACS analyses of PI-stained nuclei according to Nicoletti et al. (b)
Immunoblot analyses of Survivin expression in STA-NB15 cells after
treatment with 50 nM YM155 for the times indicated. Tubulin served as
loading control.
S8
**
Tumor weight [g]
1.50
***
1.25
ns
1.00
0.75
0.50
0.25
0.00
shSurv
shCtr
PBS
shSurv
shCtr
2DG
Supplemental Figure 8: Weight of neuroblastoma tumors from mice
treated with either diluent (PBS) or 10 mg 2DG three times a week over
21 days (n=9 animals per group). Statistical differences between PBSand 2DG-treated or control cells and Survivin knock-down cells were
assessed using Mann-Whitney U test (***P<0.001, **P<0.01).