B.
500
Tumor volume (mm3)
A.
PC3 cells
Control
VEGF165b
bevacizumab
400
Saline
100µg rhVEGF165b
bevacizumab
300
**
200
**
100
**
**
0
C.
D.
0
4
Vessels/mm-2
30
Saline
VEGF165b
8
12
Days after injection
*
**
16
25
20
15
10
5
0
Saline
VEGF165b Bevacizumab
Supplementary 1. rhVEGF165b administered intraperitoneally slows PC3 tumour growth by inhibiting
angiogenesis. A. Tumour growth in nude mice treated with saline, rhVEGF165b or Bevacizumab (tumours contours
outlined in black). B. Tumour growth curves in the three treatment groups. C. Representative pictures of tumour
sections from saline and rhVEGF165b -treated mice stained with CD31 to visualize tumour vessels. D. Quantification of
micro-vessel density in the three treatment groups (p< 0.001, One-Way Anova). * p<0.05; ** p<0.01.
20
A.
B.
SRPK1 fold expression (%)
120
100
CTRL
SRPK1
KD
80
IB: SRPK1
92kDa
IB: -actin
45kDa
60
40
20
0
control
SRPK1 KD
Supplementary Figure 2. Quantification of SRPK1 KD in stable PC3 cells transduced with
either control or SRPK1 shRNA lendiviral particles by qRT-PCR (A.) and Western blot (B.)
PAN VEGF
B
C
0.5
1.2
1
Control
KD
0.8
0.6
0.4
Control
KD
0.3
0.2
0.4
0.1
0.2
0
VEGFxxxb/VEGFxxx
0
PC3
PC3
Excess of antiangiogenic isoform
VEGF (pg/µg total protein)
A
VEGFxxxb
3
2.5
2
Control
KD
1.5
1
0.5
0
PC3
Supplementary Figure 3. SRPK1 knockdown triggers a switch in VEGF expression towards the anti-angiogenic
isoforms in PC3 as assessed by ELISA for A. Total VEGF and B. VEGFxxxb expression relative to total protein or C. Ratio
of VEGFxxxb to VEGFxxx expression. (calculated as VEGFxxxb/(VEGFtotal-VEGFxxxb)
A
CTRL shRNA
SRPK1 shRNA
SR (1H4)
SRPK1
SRSF6
SRSF5
SRSF1/2
α - Tubulin
B
mab104
SRSF4 (SRp75)
SRSF6 (SRp55)
SRSF5 (SRp40)
SRSF1/2 (ASF/SF2/ SC35)
SRSF3 (SRp20)
α - Tubulin
Supplementary Figure 4. SRPK1 knock–down alters phosphorylation of splice factors in PC-3 cells. Western
blot analysis of extracts from control and SRPK1 KD PC3 cells. A. Expression levels of SR proteins are not affected. B.
Phosphorylation levels of SRSF1, 2 and 5 are markedly decreased on SRPK1 KD cells.
900
600
400
200
Neuropilin1:
209bp
506
344
220
VEGFR1:
331bp
398
298
220
201
VEGFR2:
220bp
Supplementary Figure 5. PC-3 cells express neuropilin 1, VEGFR1 and VEGFR2. RT-PCRs
with specific primers on RNA isolated from PC3 cells and HUVECs as positive controls
SRPK1 copy no (x105
per µg RNA)
A.
5
4
3
2
1
B.
Tumour volume (mm^3)
CTRL
SRPK1 KD
2500
2000
R=0.7377
CTRL
SRPK1 KD
1500
1000
500
1
2
3
4
5
6
7
SRPK1 copy no (x105 per µg RNA)
Supplementary 6. A. SRPK1 transcript quantitation in KD and control tumours as assessed by qRT-PCR. B.
Correlation between tumour volumes and SRPK1 transcript number
0.100
Luciferase/Renilla
0.090
0.080
0.070
0.060
0.050
0.040
0.030
0.020
0.010
0.000
Wildtype
shCtl
shSRPK1
Supplementary 7. SRPK1 knockdown does not affect VEGF promoter activity. Luciferase driven by VEGF
promoter (pGL4 backbone) was transfected in either wild-type, control shRNA or SRPK1 shRNA PC3 cells together
with Renilla luciferase plasmid. No effect on VEGF promoter is seen.
SPHINXs compound validation using PC3 cell line
IP: mab104
PC3
Control
kDa
37
IB:SRSF1
SRPIN340
10uM
SPHINX
10uM
SPHINX 7
1uM
25
% Relative densitometery of
pSRSF1/ IgG band
120
100
80
60
40
20
0
control
SRPIN340
SPHINX
SPHINX7
Supplementary 8. PC3 cell were treated with SRPIN340 and SPHINXs compound for half an hour and protein extracted. The
activity of SRPK1 was determined by estimating the expression level of p-SFSR1 via immunoprecipitation (IP) with Mab104 – an
anti-phosphoSR antibody, and immunoblotting using SRSF1 antibody (Abcam)
NRP1 – AACATTCAGGACCTCTCTTGA
NRP – AGGACAGAGACTGCAAGTATGAC
VEGFR1 - AAATAAGCACACCACGC
PCR
Forward / Reverse primer
PCR
Product
Cond it ion s
size
45 cycles:
95°C for 60
s
123 &
VEGF1 65/VEGF1 65b
55°C for 60
57
s
72°C for 2 m
30 cycles:
95°C for 45
F: 5' - CACCCACTCCTCCACCTTTGAC
s
- 3'
65°C for 45
GAPDH
112
R: 5' - GTCCACCACCCTGTTGCTGTAG
s
- 3'
72°C for 60
s
F: 5’- GGCAGCTTGAGTTAAA CGA AC
G-3’
R: 5’ATGGATCCGTA TCAG TCTTTCCTG G3’
Supplementary Table 1
VEGFR1- ACCTGCTGTTTTCGATGTTTC
VEGFR2 - AAAACCTTTTGTTGCTTTTGGA
VEGFR2 - GAAATGGGATTGGTAAGGATGA