Supplementary MATERIAL AND METHODS
Immunocytochemical analyses
Quantification and analysis of stress fibers or focal adhesions were performed using ImageJ (NIH,
MA, USA) software. The cell perimeter was marked as the region of interest. The integrated densities
per area obtained from the green channel represent UTeGFP expression level. In phalloidin-stained cells,
the integrated densities per area obtained from the red channel correspond to polymerized actin. In
vinculin and integrin-labeled cells, the number and the size of focal adhesion points were calculated
using particle analysis plug-in after successive image treatment by ImageJ software.
Counting/Proliferation assay
Cells were serum-starved for 24h, then incubated in the absence or the presence of UII for 48h in
serum-free medium. Cell number was quantified by counting events with an electronic cell counter
(Z2, Beckman Coulter, Roissy CDG, France).
Cell cycle analysis
Glioma cells, serum-starved for 24h, were incubated in the absence or the presence of UII for 24h.
Cell cycle analysis was done by DNA staining with propidium iodide (PI, Sigma-Aldrich) by flow
cytometry. Briefly, after treatment, cells were fixed in ice cold 70% ethanol for 15 (SW1088) or 30
min (U87). Staining was done with 0.6 mg/ml RNase (RT, 30 min) and 50 µg/mL PI (RT, 30 min).
Cell cycle status was quantified by FACScalibur flow cytometer using CellQuest analysis software.
Calcium measurement
Cells grown into 96-well plates were rinsed twice with a modified HBSS (135 mM NaCl, 5 mM KCl,
1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, 3 mM glucose and 2.5 mM probenicid), and incubated
with 2 μM Fluo-4 AM dye (Molecular Probes) containing 20% pluronic acid for 1h. The effects of
ATP 0.1 µM or graded concentrations of UII on [Ca2+]c were measured with a fluorimetric imaging
plate reader FlexStation III (Molecular Devices, Wokingham, England) using the SoftMaxPro 5
software.
For Ca2+ microfluorimetry on fresh glioma explants, intracellular Ca2+ was measured (fluorescence at
510 nm following excitation at 340/380 nm from a 300 W xenon lamp) from a single visual field using
an integrating CCD (charge-coupled device) camera coupled to a Leica DMLFSA microscope. Cell
Ca2+ imaging and fluorescence quantification were carried out using the Metafluor Imaging System
(Molecular Devices).
Wound healing assay
U87 cells (500,000/well) were plated in a fibronectin-coated 12-well plate. After seeding, cell
monolayer was scratched with a tip, and UII was added. Microphotographies of the wound were taken
at 0h and 16h by using 4X objective of a microscope (Nikon Eclipse TS 1000).
cAMP/IP-1 metabolism
The determination of the inositol phosphate 1 (IP1) accumulation in HEK293 cells was performed in
96-wells microplates using the IP-One HTRF® assay (Cisbio Bioassays, Codolet, France) according
to the manufacturer’s protocol. Time-resolved fluorescence resonance energy transfer (FRET) signals
were measured 50 μs after excitation at 620 and 665 nm using a RubyStar instrument (BMG labtech,
Champigny-sur-Marne, France).
Cell-based ELISA
Cells (50,000/well) were plated in poly-ornithine-coated 96-well plates. After fixation with PFA (4%,
5 min, RT), cells were permeabilized in 0.05% Triton X-100 and non specific binding sites were
blocked with FBS (1%, 30 min, RT). Cells were incubated with anti-c-myc antibody (0.5 µg/ml,
Santa-Cruz) 1h at RT, then with HRP-conjugated secondary antibodies (1/1000, Santa-Cruz) for 30
min at RT. After washes, a colorimetric alkaline phosphatase substrate was added (SuperSignal
ELISA, Fisher Scientific) and the resulting color reaction was measured using a luminometer (Fisher
Scientific).
Phosphospecific cell-based ELISA was performed as previously described with minor
modifications (Versteeg et al. 2000). Primary antibodies directed against ERK and p-ERK (Santa
Cruz), or AKT and p-AKT (Upstate) were used at 0.5µg/mL each. Luminescence was measured using
a luminometer (Fisher Scientific) after addition of a colorimetric alkaline phosphatase substrate
(SuperSignal ELISA, Fisher Scientific).
Cloning ring assay
HEK293 cells were plated in a cloning ring 6 mm diameter (50,000/ring). After seeding, the ring was
removed, cells were rinsed with fresh serum-free medium and UII was added. Microphotographies of
the cell ring were taken at 0h, 24h and 48h by using a Macroscope Leica (Z6 APO) and the cell disc
expansion was quantified by subtracting the area corresponding to the initial cellular edge at 0h from
that of the edge of the cell disc after 24h or 48h treatment by using ImageJ software.
Supplementary Legends to figures
Figure S1. Clinicopathologic features of the studied glioblastoma. GBM, glioblastoma
multiform; M, male; WHO, World Health Organization. NA, not analyzable because of large
necrotic areas.
Figure S2. Validation of the antibody directed against human UT in HEK cells. Left, Western
blot analysis of UTcmyc expression using the anti-UT antibody used in Figure 1D in HEK
transfected with c-myc or transfected with human recombinant UTc-myc (UT). A Specific band
and a higher molecular mass doublet of bands likely corresponding to human UT appeared at
≈42 and around 65 kDa (black arrows). Co-transfection of HEK-UTcmyc with shRNA directed
against UT mRNA (shRNA UT) showed a slight diminution of UT expression at ≈ 45 kDa,
not observed after transfection with a control shRNA (shRNA Ctrl). Right, Expression of
UTc-myc in whole cells (permeabilized) or only at the cell plasma membrane (nonpermeabilized) using anti-UT antibody and revealed by ELISA assay.
Figure S3. Effect of UII on glioma cell proliferation, cell cycle and chemotaxic migration.
(a) SW1088, U87 and HEK-UT were incubated for 48h in the absence or the presence of UII
(10-8 and 10-10 M) or FBS and counted using a Coulter counter. UII did not modify cell
growth whereas FBS exhibited drastic stimulation of cell proliferation/survival. Data are
mean  SEM from one representative experiment in quadruplicate of 4 independent
experiments. Mann and Whitney test, *, P<0.05; compared with the corresponding control (in
the absence of treatment). (b) Representative example of the UII (10-7 M) effect on SW1088
and U87 cell cycles after a 24h-treatment. Cell cycle analysis following propidium iodide
intercalation into the cellular chromatin was performed by flow cytometry. Data are
represented as relative fluorescence intensity of G1, S and G2/M-phase population in a 2dimensional cytometry profile in the absence (control) or the presence of UII. Percentage cells
in each phase are indicated. (c) SW1088 (left) and U87 (right) were exposed in the Boyden
chamber to increasing concentrations of UII (10-13-10-7 M) for 24h and cells were counted as
described above. Bar graph represent mean  SEM obtained from 3 independent experiments
in triplicates (SW1088) or from a representative experiment in triplicates (U87). One way
analysis of variance followed by a Bonferroni post-test, *, P<0.05; **; P<0.01, ***<0.001. (d)
Wound healing assay showing in vitro directional migration of U87 after a scratch of a
confluent cell layer in the absence (FBS-) or the presence of FBS (FBS+) and UII (10-10 and
10-8 M). Photographs were taken at 0h and 16h after scratch injury and showed that UII (10-10
and 10-8 M) enhanced the covered surface compared to control (FBS-).
Figure S4. Glioma cells do not exhibit the classical UT/Ca2+ coupling. (a) Left,
concentration-response curve for UII (10-12-10-7 M)-mediated [Ca2+]c mobilization in HEKtransfected with cDNA encoding c-myc (HEK) or UTc-myc (HEK-UT). Ca2+ response was
recorded from cells previously loaded with Fura2-AM, and ATP (10-4 M) was used as a
positive control. Middle, panel shows the quantification of the maximum Ca2+ response
detected at 10-7 M (UII) and 10-4 M (ATP) normalized to control (buffer injection). Right,
concentration-response curve for UII-mediated IP1 production (normalized to the maximum
effect of UII (10-5 M). (b) Left, concentration-response curve for UII (10-12-10-7 M)-mediated
[Ca2+]c mobilization in glioma SW1088 and U87 cells transfected with cDNA encoding cmyc (SW1088 and U87) or UTc-myc (SW1088-UT and U87-UT). Right, quantification of the
maximum effect of UII (10-7 M) and ATP (10-4 M) on [Ca2+]c mobilization in glioma cells. (a
and b) Data are mean  SEM from 2 experiments in triplicates. Statistical significance was
determined by a Kruskal-Wallis test followed by a Dunn's Multiple Comparison Test. *,
P<0.05; **, P<0.01; ***, P<0.001 compared with control. (c) Left, Pseudocolor images
illustrating time-dependent increase in [Ca2+]c in a representative experiment on a human
glioblastoma explant (72h of culture) following exposure to UII (10-7 M). (a) glioma cells
under resting conditions in a single visual field (bar correlates pseudocolor with relative
[Ca2+]c levels). (b) a subset of glioma cells non responding upon addition of UII (10-8 M, 150
s). (c) ATP (10-4 M, 40 s)-inducing a rapid (c) and reversible (d) [Ca2+]c increase in the same
glioblastoma explant. Right, composite of [Ca2+]c mobilization responses in 16 individual
cells from the microscopic field in the left. All cells failed to respond to UII and exhibited a
marked Ca2+ rise after addition of ATP. Scale bar, 50 µm.
Figure S5. Control experiments on UTERY(AAA) mutant, coupling to Go and G12 proteins in
HEK cells. (a) Western blot analysis showing expression of UTc-myc (HEK-UT) and the
UTERY(AAA)c-myc mutant (HEK- UTERY(AAA)) revealed with an anti-c-myc after 48h transient
transfection. A triplet band was detected for UT and UTERY(AAA) at ≈40, 45 and 47 kDa and
also a thin band around 65 kDa, compared with HEK. (b) Expression of UTc-myc and
UTERY(AAA)c-myc mutant expressed as receptors in whole cells (permeabilized) or only at the
cell plasma membrane (non-permeabilized) using anti-c-myc antibody and revealed by
ELISA assay. The UTERY(AAA) mutant was significantly expressed at the plasma membrane
and showed similar expression than UT. Bar graph represents mean  SEM from 2
experiments in sextuplet. Statistical significance was determined by a Mann and Whitney test,
**, P<0.01; ***, P<0.001. (c) HEK were transiently transfected with cDNA encoded pCMVcmyc
, UTc-myc or UTERY(AAA)c-myc and incubated with the Ca2+ fluorescent probe Fluo 4.
Application of UII (10-7 M) induced a maximum amplitude of [Ca2+]c transients expressed as
percentage of the corresponding control values in the absence of UII. Data are mean  SEM
from a representative experiment in triplicate. (d) Assessment of BRET between Gαo and Gβγ
or UT and Gα12 in HEK co-expressing αo-Rluc and βγ-eYFP or UT-RlucII and α12-eGFP2 in
the presence of increasing concentrations of UII (10-11 to 10-6 M).
Figure S6. Urotensin II effect on ERK1/2 and AKT phosphorylation in HEK-UT and glioma
cells. Determination of both total and phosporylated (activated) forms of ERK1/2 (a, c, e) and
AKT (b, d, f) on HEK expressing UT (HEK-UT, a and b), SW1088 (c and d) and U87 (e and
f) in the absence (control) or the presence of UII from 0.5 to 30 min. FBS and/or EGF were
used as positive controls whereas an inhibitor of MAPK (PD98059) was used as a negative
control for ERK1/2 activation. ELISA assay was performed with specific antibodies directed
against total and phosphoforms of ERK1/2 and AKT, respectively, as described in Materials
and Methods. Bar graph represents mean  SEM from 2 to 3 experiments in triplicates. For
time course, statistical significance was determined by a Kruskal-Wallis test followed by a
Dunn's Multiple comparison Test. *, P<0.05; **, P<0.01; ***, P<0.001 compared with
control. For positive or negative controls, statistical significance was determined by a Mann
and Whitney comparison Test. *, P<0.05; **, P<0.01; ***, P<0.001 compared with control.
Figure S7 Inhibition of HEK-UT cell motility in the cloning ring assay. (a) Monolayer
disc of HEK transfected with cDNA encoding UT were formed in cloning ring during 24h in
fibronectin, and then were incubated in the absence (control) or the presence of UII (10-14 to
10-6 M) during 48h. Top panel indicates a representative phase contrast photography of
spheroid morphology at 0 (small dotted white line) and 48h (large dotted white line). The
bottom panel represents the quantification of monolayer disc outgrown corresponding to the
disc surface area measured at 48h normalized to its area at T0. Administration of UII
drastically blocked cell expansion. (b) Top panel, photographs of monolayer disc of HEK
expression UTeYFP (green cells) acquired from time-lapse microscopy over a 14h-imaging
period. Bottom panel, motility of border cells non expressing (UT-) or expressing (UT+) UT,
in the absence (control) or the presence of UII (10-8 M) during 8h. It can be noticed that UTpositive cells covered less distance even in the absence of UII than UT negative cells.