6
5
4
3
2
Cell no x 10000
7 kDa
6
5
4
3
2
1
RAN
25
β-Actin
47
R37-OPNoligo 1,3
R37-OPNoligo 2,3
R37-OPNoligo 1,2
R37-OPNoligo 3
R37-OPNoligo 2
R37-OPNoligo 1
R37OPN/siRNARAN
R37-OPN
200
180
160
140
120
100
80
60
40
20
0
Colony no
Supplementary data
Figure 1
Fig 1A
Fig 1B
8
7
1
0
Fig 1C.
R37-OPNoligo 1,3
R37-OPNoligo 2,3
R37-OPNoligo 1,2
R37-OPNoligo 3
R37-OPNoligo 2
R37-OPNoligo 1
R37OPN/siRNARAN
R37-OPN
Fold Induction
Fig.1D
1.4
1.2
1
0.8
0.6
0.4
0.2
0
pM
1
VP
p
+
n
6
pM
-O
Ra
61
P
pV
+
PN
+
n
Ra
61
P
pV
pM
VP
+p
16
PN
-O
pM
Fig.1 The expression and biological effect of each oligonucleotide combination to RAN from the siRNA
library
A Immunoblot for RAN protein with R37-OPN cells transfected with individual siRNA-RAN
oligonucleotides. Cell lysates were diluted and 5µg loaded onto a SDS 10% (w/w) polyacrylamide gel, as
follows: lane 1 R37-OPN; lane 2 R37-OPN-oligo 1; lane 3 R37-OPN-oligo 2; lane 4 R37-OPN-oligo 3; lane
5 R37-OPN-oligo 1,2; lane 6 R37-OPN-oligo 2,3; and lane 7 R37-OPN-oligo 1,3. Specific proteins were
detected using antibodies to RAN or -actin. Bands were quantified using densitometric analysis and
normalised with respect to those of β-actin. The results were a representative sample of three experiments.
There was no change in the level of OPN protein. B The ability of stably transfected cell lines to adhere to a
laminin treated surface was assessed over a 30 min period and the number of adherent cells quantified. All
three oligonucleotides from the siRNA RAN mixture were stably transfected separately into the R37-OPN cell
line. The results were the mean ± standard error of three independent experiments.
C Soft agar assays were carried out to asses the ability of stably transfected cell lines to grow in an anchorageindependent environment. The colony number was assessed after 5 days. The results were the mean ±
standard error of three independent experiments.
The expression of three separate siRNA oligonucleotides targeted against RAN inhibits the three properties
associated with the malignant, metastatic state in R37-OPN cells to varying degrees. This result is consistent
with RAN being a downstream effector of the same malignant, metastatic state induced by OPN. This
conclusion is confirmed by the similar incremental decreases in expression of RAN protein (Fig. 1A) and in
cell adhesion (Fig. 1B) and anchorage-independent growth (Fig. 1C) when the R37-OPN cells are transfected
singly or in binary combinations with the three siRNA oligonucleotides targeted against RAN.
D Mammalian two-hybrid assays to detect interactions of OPN with Ran. Rama 37 cells were transiently cotransfected using pVP16-Ran or pVP16 plasmid with pM-OPN or pM plasmid. A reporter plasmid pG5-CAT
was included in all co-transfections. All co-transfections also contained pSV40-β-Galactosidase expression
vector, which worked as a β-galactosidase internal control reporter to normalize for transfection efficiency.
Forty-eight hours after transfection, the transfected cells were collected and CAT enzyme assay was
performed. Fold induction was performed compared to the cells cotransfected with empty pM and empty
pVP16 vector for all the CAT assay result. Experiments were performed in triplicate. Values are means with
S.D. from triplicate independent determinations. * p>0.05 compared to cell cotransfected with pM-OPN and
pVP16-Ran.