Electronic supplementary material consisting of: legends to
supplementary figures, supplementary materials and methods
BMP-7 inhibits TGF--induced invasion of breast cancer cells through
inhibition of integrin 3 expression
Cellular Oncology
Hildegonda P.H. Naber1,*, Eliza Wiercinska1,*, Evangelia Pardali1, Theo van Laar1,
Ella Nirmala2, Anders Sundqvist4, Hans van Dam1, Geertje van der Horst3, Gabri
van der Pluijm3, Bertrand Heckmann5, Erik H.J. Danen2 and Peter ten Dijke1,4§
1
Department of Molecular Cell Biology and Centre for Biomedical Genetics, Leiden
University Medical Center, P.O. box 9600, 2300 RC, Leiden, The Netherlands, 2Division
of Toxicology, LACDR Leiden University, Gorlaeus Laboratories, Einsteinweg 55, 2300
RA Leiden, The Netherlands, 3Dept. of Urology and Endocrinology, Leiden University
Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands, 4Ludwig Institute
for Cancer Research and Uppsala University, Box 595, 75124 Uppsala, Sweden,
5
Galapagos SASU, 102 Avenue Gaston Roussel 93230, Romainville, France
*these authors contributed equally
§
To whom correspondence should be addressed: : p.ten_dijke@lumc.nl
Supplementary figures
Supplementary Fig. 1 BMP-7 does not affect cell growth. Cell viability of M-IV was
measured after 0, 24 and 48 hr of treatment with no ligand, BMP-7 (100 ng/ml), TGF-
(5 ng/ml) or TGF- & BMP-7
M-IV
relative growth
5
BMP-7
TGF-
TGF- & BMP-7
4
3
2
1
0
0
1
2
time (days)
Supplementary Figure 2
No effect of Noggin on basal invasion of M-II. Spheroids of M-II were allowed to
invade collagen in the absence or presence of Noggin (200 ng/ml). (a) Pictures of
representative spheroids. (b) Quantification of spheroids of experiments shown in (a).
The results are expressed as mean ± S.D. of at least three spheroids per condition. One
representative out of three independent experiments is shown.
Supplementary Figure 3
BMP-7 does not inhibit TGF--induced integrin v3 expression in M-II. Spheroids were
embedded with BMP-7 (100 ng/ml), TGF- (5 ng/ml), TGF- & BMP-6 or TGF- &
BMP-7 for 24 hr. Integrin v (a) and integrin 3 (b) mRNA expression was analyzed by
Q-PCR . One representative out of three independent experiments is shown.
Supplementary Table 1
Specificity of GLPG0187 towards integrins. GLPG0187 was profiled in a solid phase
assay for its ability to inhibit integrin-ligand interactions for integrins v1, v3, v5,
v6, v8, 51, 21, 47 and IIb3. The table summarizes IC50 values for GLPG0187
in nM. Data are expressed as mean ± S.E.M. from three independent experiments
GLPG0187
v1
v3
v5
1.3 ±
3.7 ±
2.0 ±
0.1
0.6
0.6
v6
v8
51
1.4 ± 0.3
1.2 ± 0.3
7.7 ± 4.0
2
1
4
7
>1
>1
4
4
0
0
IIb
3
>105
Supplementary Fig. 4 Confirmation of knockdown and overexpression of integrin 3. (a)
M-IV cells were transduced with control lentivirus or a mixture of lentivirus encoding
shRNA against integrin 3 and selected with puromycin. Cells were treated with or
without TGF- (5 n/gml) for 24 hr before RNA was isolated and analyzed by Q-PCR for
integrin 3 expression. (b) M-IV cells were transduced with lentivirus encoding integrin
3 and subjected to FACS analysis
Supplementary material & methods
Cell proliferation
Cells were seeded at a density of 5x102 cells/well in 96-well plates. The next day,
medium was refreshed and TGF-β was added. Cell number was determined at days 0, 1, 2
and 3 by adding MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4sulfophenyl)-2H-tetrazolium, inner salt, Promega), followed by measuring the absorbance
at 490 nm.
Integrins and integrin ligands
Integrin 21 was purified from detergent extracts of HT1080 fibrosarcoma cells as
previously described [1]. Recombinant integrin 47 was purchased from R&D systems
(Oxford, UK). Recombinant integrin 51 was made as an Fc fusion protein as described
before [2]. Recombinant v integrins were made using a baculovirus expression system in
insect cells.
Rat tail tendon collagen was obtained from Sigma-Aldrich (Poole, Dorset, UK) and
biotinylated as described [1]. Recombinant murine MAdCAM-1 Fc fusion protein was
made in COS cells. A recombinant fragment of human fibronectin containing type III
repeats 6-10 was produced and biotinylated as described [3]. Purified human vitronectin
was obtained from Sigma-Aldrich. Recombinant human LAP (latency associated protein)
was purchased from R&D systems, and biotinylated using sulfo-NHS-LC-biotin (Pierce)
according to the manufacturer’s protocols.
Solid phase assay
For solid phase assays the binding of ligands to integrins was measured in the presence of
1mM Mn2+ to activate the receptors. In brief, 96-well were coated with integrins (1-5
µg/ml in Dulbecco’s PBS) overnight at room temperature. Wells were then blocked for 13 hr with 5% (w/v) BSA, 150 mM NaCl, 0.05% (w/v) NaN3, 25 mM Tris-Cl, pH 7.4.
Wells were then washed three times with 200 µl of 150 mM NaCl, 25 mM Tris-Cl, 1 mM
MnCl2, pH 7.4, containing 1 mg/ml BSA (buffer A). Ligands in buffer A were added to
the wells in the absence or presence of inhibitors, and the plate was then incubated at
room temperature for 2 hr. The wells were washed three times with buffer A. For
biotinylated ligands, bound ligand was quantitated by addition of 1:500 dilution of
ExtrAvidin peroxidase conjugate (Sigma-Aldrich) in buffer A for 30 min at room
temperature. For MAdCAM-Fc, bound ligand was quantitated by addition of 1:1000
dilution of anti-human Fc peroxidase conjugate (Jackson) 30 min at room temperature.
For vitronectin, bound ligand was quantitated by addition of 1:500 dilution of anti-human
vitronectin antibody VIT-2 (Sigma-Aldrich) for 30 min at room temperature followed by
1:1000 dilution of anti-human IgM peroxidase conjugate (Jackson) for 30 min at room
temperature. Wells were then washed four times with buffer A and the assay was
developed
using
ABTS
(2,2'-Azinobis
[3-ethylbenzothiazoline-6-sulfonic
acid]-
diammonium salt), followed by measuring absorbance at 410 nm. Background binding of
ligands to wells coated with BSA alone was subtracted from all measurements. IC50=
concentration of inhibitor for 50% of maximal inhibition of binding of ligand binding to
the integrin used in the assay.
FACS analysis
Cells were detached by EDTA, filtered, washed and incubated with anti-integrin 3
antibody (Abcam) or no antibody in 2% FBS/PBS for 1 hr at 4 C. Cells were washed and
incubated with anti-mouse-FITC (DAKO) for 30 minutes at 4C and washed. Surface
expression was measured on a FACSCanto (B&D).
Supplementary references
1. Tuckwell DS, Smith L, Korda M, Askari JA, Santoso S, Barnes MJ, Farndale RW,
Humphries MJ. Monoclonal antibodies identify residues 199-216 of the integrin 2
vWFA domain as a functionally important region within 21. Biochem J. 2000;350 Pt
2:485-493.
2. Coe AP, Askari JA, Kline AD, Robinson MK, Kirby H, Stephens PE, Humphries MJ.
Generation of a minimal 51 integrin-Fc fragment. J Biol Chem. 2001;276:3585435866.
3. Mould AP, Askari JA, Aota S, Yamada KM, Irie A, Takada Y, Mardon HJ, Humphries
MJ. Defining the topology of integrin 51-fibronectin interactions using inhibitory
anti-5 and anti-1 monoclonal antibodies. Evidence that the synergy sequence of
fibronectin is recognized by the amino-terminal repeats of the 5 subunit. J Biol Chem.
1997;272:17283-17292.