Supplementary Information Persistent signaling induced by ... · Supplementary Information ....

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Supplementary Information Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors Florian Mullershausen 1, Frédéric Zecri 2 , Cihan Cetin 4 , Andreas Billich 3 , Danilo Guerini 3 and Klaus Seuwen 1Novartis Institutes for BioMedical Research, 1 Developmental & Molecular Pathways, 2 Global Discovery Chemistry, 3 Autoimmunity, Transplantation & Inflammation, Novartis Pharma AG, Basel, Switzerland. 4 Friedrich Miescher Institute for Biomedical Research, Facility for Advanced Microscopy and Imaging, Basel, Switzerland To whom correspondence should be addressed: Florian Mullershausen or Klaus Seuwen Novartis Pharma AG Forum 1 Novartis Campus CH-4056 Basel Tel. +41 61 324 2910 Fax. +41 61 324 6105 [email protected] [email protected] 1 Nature Chemical Biology: doi: 10.1038/nchembio.173

Transcript of Supplementary Information Persistent signaling induced by ... · Supplementary Information ....

Page 1: Supplementary Information Persistent signaling induced by ... · Supplementary Information . Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors

Supplementary Information

Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors

Florian Mullershausen1¶, Frédéric Zecri2, Cihan Cetin4, Andreas Billich3, Danilo Guerini3 and Klaus Seuwen1¶

Novartis Institutes for BioMedical Research, 1Developmental & Molecular Pathways, 2Global Discovery Chemistry, 3Autoimmunity, Transplantation & Inflammation, Novartis Pharma AG, Basel, Switzerland. 4Friedrich Miescher Institute for Biomedical Research, Facility for Advanced Microscopy and Imaging, Basel, Switzerland ¶To whom correspondence should be addressed: Florian Mullershausen or Klaus Seuwen Novartis Pharma AG Forum 1 Novartis Campus CH-4056 Basel Tel. +41 61 324 2910 Fax. +41 61 324 6105 [email protected]@novartis.com

1Nature Chemical Biology: doi: 10.1038/nchembio.173

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Supplementary Figures

a

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- -- FTYP

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FTYP FTYP- FTYP

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FTYP FTYP- FTYP

- -- FTYP

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Supplementary Figure 1: Control experiments using CHO-S1P1 and CHO-S1P3 cells (a) The inhibition of Fsk-induced formation of cAMP was measured in CHO-S1P1 cells in

the presence of the indicated concentrations of S1P or FTY720P. The bar graph shows

the responses to Fsk in PTX-treated cells in the absence or presence of maximally

effective concentrations of S1P or FTY720P, shown is a representative experiment. (b)

CHO-S1P3 cells were preincubated without or with FTY720P (1 µM) or S1P (1 µM) for 1

h, washed, and further incubated in the absence of agonists for 5 h. Then, Fsk-induced

cAMP formation was assayed in the absence of presence of FTY720P (10 nM). Data

represent mean ± SEM of ≥ 3 independent experiments.

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40

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FTYP (nM) during preinc.

cAM

P (%

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sk)

5h washout24h washout

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FTYP (nM) during preinc.

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FTYP FTYP10 nM 1 µM

control

PTX

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sk c

ontro

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bFsk

Supplementary Figure 2: Reversibility of persistent signaling and Gi-protein-mediated sensitization of cAMP formation (a) Fsk-induced cAMP formation in CHO-S1P1 cells that were preincubated without or

with FTY720P at the indicated concentrations for 1 h, washed, and further incubated in

the absence of agonists for 5, 24 or 48 h. (b) CHO-S1P1 cells were preincubated without

or with the indicated concentrations of FTY720P (1 h), washed and further incubated for

5 h with or without PTX in the absence of agonists. Then, Fsk-induced cAMP formation

was determined. Data represent mean ± SEM of ≥ 3 independent experiments.

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a

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Supplementary Figure 3: Quantification of S1P1 receptor distribution in CHO-S1P1 cells (a) CHO-S1P1 cells expressing an N-terminal extracellular myc-epitope tag were

stimulated with the indicated agonist (1 µM, 1h) and stained with an anti-myc antibody

and a secondary antibody labeled with Alexa 488 fluorescent dye. 10,000 viable cells in

each sample were analyzed using flow cytometry. (b) Cell surface localization of S1P1

as detected by flow cytometry expressed as geometric mean ± SEM derived from ≥ 3

independent experiments. (c) Images of CHO-S1P1 cells treated with the indicated

agonists (1 µM, 1h) and stained with anti-S1P1 antibodies were analyzed as described

in the Supplementary Methods section. Data are expressed as average Mahalanobis

Distance from FTYP-treated cells and were derived from 40 – 60 single cells.

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a

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Supplementary Figure 4: Characterization of S1P receptor-mediated signal transduction in HUVECs (a) Inhibition of Fsk-induced cAMP formation in HUVECs in the presence of the indicated

concentrations of FTY720P or S1P. Where indicated, the antagonist W146 (10 µM) was

added to the cells 15 min prior to the assay. (b) Western blot detection of pERK after

stimulation of HUVECs with the indicated concentrations of FTY720P or S1P. Where

indicated, W146 (10 µM) was added to the cells 25 min prior to stimulation. Calnexin

was detected to control for protein content of the samples. (c) HUVECs were

preincubated without or with the indicated FTY720P (1 µM, 1 h), washed and further

incubated for 4 h with or without PTX in the absence of agonists. Then, Fsk-induced

cAMP formation was determined.

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0

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S1P S1P- FTYP

FTYP FTYP- FTYP

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Preinc.:Assay:

cAM

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Fsk Supplementary Figure 5: Persistent signaling of FTY720P in primary astrocytes Primary rat astrocytes were preincubated without or with FTY720P (1 µM) or S1P (1 µM)

for 1 h, washed, and further incubated in the absence of agonists for 5 h. Then, Fsk-

induced cAMP formation was assayed in the absence of presence of FTY720P (10 nM).

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GM130 LAMP1

1 h

3 h

6 h

8 h

Supplementary Figure 6: Localization of S1P1 in HUVEC after long-term incubation with FTY720P Co-immunostaining of S1P1 receptors (green) and the indicated organelle markers (red)

in HUVECs stimulated with FTY720P (1 µM) for 1, 3, 6 or 8 h. Left panel shows GM130

(Golgi matrix), right panel shows LAMP1 (lysosomes). Scale bar: 10 µm

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Supplementary Methods

Antibodies and Reagents

S1P was purchased from Biomol (USA) and dissolved in DMSO containing 50 mM HCl

to yield a stock solution of 5 mM. Rabbit polyclonal antibodies to human S1P1 receptor

were from Affinity BioReagents (USA) and from Santa Cruz Biotechnology (USA).

Mouse monoclonal antibodies against organelle resident antigens EEA1, CD107a,

GM130, p230 were from BD Biosciences (USA), mouse monoclonal antibodies against

clathrin and Mannose-6-phosphate receptor were from Abcam (UK). For Western blot

detection, antibody against phosphorylated ERK was from Cell Signaling (USA),

Calnexin antibody was from BD Biosciences (USA) and βActin antibody was from Sigma

(Switzerland).

In vitro phosphorylation of FTY720 and OC5

The phosphorylation reactions were performed essentially as described1. Briefly, the

cytoplasmic fraction of recombinant HEK-293 cells overexpressing human SPHK-2 was

incubated at 30°C in a total volume of 100 μL with FTY720 or OC5 (20 μM; added from

stock solutions in DMSO), 1 mM of ATP, and 2 μCi [γ-32P]ATP in 50 mM Hepes buffer

(pH 7.4) containing 15 mM MgCl2, 0.005% Triton X-100, 10 mM KCl, 10 mM NaF and

1.5 mM semicarbazide. Following incubation for different time points up to 2 h, lipids

were extracted and separated by thin-layer chromotography. Radiolabeled FTY720P

derivatives were visualized and quantified using a Molecular Dynamics Storm

PhosphorImager (Sunnyvale, CA). From the data, the rate of phosphorylation was

calculated as nmoles of product formed per min per mg of total protein.

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Flow cytometric analysis of surface expression of S1P1 upon agonist treatment

CHO-S1P1 cells were grown in six-well plates to 60-80% confluence in αMEM, 10 %

charcoal-stripped lipid-depleted FBS, 50 µg/ml Gentamicin. The cells were incubated for

1h with a 1 μM agonist solution at 37°C in a CO2 incubator in complete medium. After

the incubation and treatment with the compounds the cells were washed with

phosphate-buffered saline (PBS) and detached by treatment with PBS/1 mM EDTA. 106

cells were incubated either with 4 μg/ml of monoclonal mouse anti c-myc IgG1 (Roche

Applied Science, Switzeraland) antibody or with isotype control mouse IgG1

(Pharmingen, BD Biosciences, Switzerland) in balanced salt solution (BSS)–5% FCS

(BSS: 0.14 M NaCl, 1 mM CaCl2, 5.4 mM KCl, 0.8 mM MgSO4, 0.3 mM NaH2PO4, and

0.4 mM KH2PO4 [pH 6.9]) for 60 min at 4ºC. The cells were washed once in BSS/5%

FCS, and incubated 60 min at 4ºC in the dark with 1 μg/ml of Alexa488-labeled goat anti

mouse secondary conjugates (Invitrogen, Switzerland). The cells were washed again

before being subjected to cytofluorometric analysis (FACS Calibur, BD Biosciences,

Switzerland). Flow cytometric measurements were performed with 10,000 viable cells

per sample. The viability was tested by the addition of 3 μl of a 1 μM DNA binding TO-

PRO-3 iodide solution (Invitrogen, Switzerland) and analyzed by flow cytometry.

Immunocytochemistry

Cells were seeded into 8-well chambered coverglasses (Lab-Tek II, NUNC, USA) and

grown for 2-3 days. 1 h prior to treatment, the growth medium was replaced by FCS-free

medium. After treatment, we fixed and permeabilized the cells using the

Cytofix/Cytoperm kit (BD Biosciences, USA). Chambers were washed with PBS,

Cytofix/Cytoperm solution was added (300 µl/well) and the slides were put on 4°C for 20

min. The wells were then rinsed twice with washing solution (1x, diluted from 10x stock;

BD Biosciences) and blocking buffer (140 mM NaCl, 10 mM Hepes, 5 mM CaCl2, 5%

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FCS, 1% BSA, 0.04% NaN3 in washing solution) was added (300 µl/well) for at least 20

min at room temperature. Primary antibodies were diluted into blocking buffer and left on

the samples over night at 4°C. Cells were rinsed 2x and incubated with anti-rabbit and/or

anti mouse secondary antibodies labeled with Alexa 488 or Alexa 633 fluorescent dyes

(Invitrogen, Switzerland) for 2-3 h, washed and viewed with a Zeiss LSM 510 META

confocal microscope (Carl Zeiss AG, Germany) using 63x oil immersion (NA 1.4) or 63x

water immersion (NA 1.2) objectives; nuclei were stained with DAPI. Images were

acquired in multi-scan mode, i.e. all colors were scanned sequentially. In co-localization

studies, optical sections were recorded at a thickness of 1.2 µm for all color channels.

Image Analysis

Images were analyzed using the ImageJ software in order to quantify differences in

receptor localization after treatment of cells with the different compounds. Prior to the

analysis, a total of 40-60 cells per experimental condition was manually defined. A

customized plugin was implemented to analyse differences using Haralick texture

analysis2, Granulometry3 and Level-set4 methods. 22 features were measured for every

cell in every field of view, most of which were highly correlated. Principal Component

Analysis5 was used to uncorrelate the features obtained in the previous operation thus

reducing the number of dimensions from 22 to 13. Next, a covariance matrix of 13 non-

correlated features was generated based on all FTY720P-treated cells, which was then

used to calculate the Mahalanobis Distance6 of each single cell (of every treatment) from

the center of mass of FTY720P-treated cells. The Mahalanobis Distance has the

advantage of being scale-invariant compared to the Euclidian distance, so that features

with high values will not be weighed to a greater extent compared to a feature with low

values.

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Western blot detection of phosphorylated ERK (pERK)

For Western blot detection of pERK, cells were cultured in 6-well plates. Starvation time

of cells in FCS-free medium was 4-5 hours prior to simulation. When indicated,

preincubations were carried out as described below. To induce ERK phosphorylation,

FTY720P was added at a final concentration of 10 nM and cells were incubated for 10

min at 37°C. Reactions were terminated by the addition of Laemmli sample buffer (Bio-

Rad, Switzerland) and boiling of the samples for 5 min. SDS-PAGE was carried out

using the Criterion system with 4-16% Tris-HCl gradient gels (Bio-Rad, Switzerland),

followed by electrotransfer of proteins onto nitrocellulose membranes. Incubation of the

membranes with primary antibodies was carried out over night at 4°C, for the control of

protein content of the samples antibodies against βActin (CHO-S1P1) or Calnexin

(HUVEC) were used. Appropriate secondary antibodies conjugated with horseradish

peroxidase (Pierce, USA) were applied followed by chemiluminescent detection of bands

using the SuperSignal™ West Dura detection kit (Pierce, USA). Visualization was

performed with a Chemidoc XRS system and the Quantity 1 software (Bio-Rad,

Switzerland).

Scratch-wound assay for migration

HUVECs were plated in 8-well µ-Slides with the ibiTreat surface (ibidi, Germany) and

grown to confluence. The growth medium was exchanged with basal endothelial medium

(without supplement, Promocell) supplemented with 1% charcoal-stripped FCS and

equilibrated for 4 h at 37°C. Pretreatments were carried out as above, by adding

FTY720P or S1P (1 µM) or control buffer and incubation of the cells for 60 min at 37°C.

Then, cells were washed 3x with PBS supplemented with CaCl2 (0.9 mM) and MgCl2

(0.5 mM). Cross-shaped scratches were introduced into each well using a 1000 µl sterile

plastic pipet tip (Eppendorf, Germany) and cells were washed again 2x to remove

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debris. Cells were allowed to migrate in basal endothelial medium (1% charcoal-stripped

FCS) for 24 h. As a positive control, FTY720P (10 nM) was included in the medium

during the migration period. A picture of the same microscopic field was taken at the

beginning and the end of the migration period, using an Axiovert 200M microscope with

a 10x objective (Carl Zeiss AG, Germany). For analysis, the migration fronts were

defined and the distances were measured using the Zeiss LSM510 standard imaging

software (Carl Zeiss AG, Germany). The distance migrated was calculated by

subtracting the distance of the migration fronts measured at the beginning of the

experiment from the distance measured after the 24 h migration period. For each

experimental condition, 4 wells were used to obtain the mean value in every experiment.

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Supplementary references

1 Billich, A. et al. Phosphorylation of the immunomodulatory drug FTY720 by

sphingosine kinases. J. Biol. Chem. 278, 47408-47415 (2003).

2 Haralick, R.M. Shanmugam, K. & Dinstein, I. Textural features for image

classification. IEEE Trans. on Systems, Man, and Cybernetics 3(6), 610–621

(1973).

2 Soille, P. Morphological Image Analysis: Principles and Applications, 2nd edition

(Springer Verlag, New York, 2003).

4 Osher, S. & Fedkiw, R. LSM: An overview and some recent results. UCLA IPAM

reports, 1-65 (2001).

5 Huang, K., Velliste, M. & Murphy, R. F. Feature Reduction for Improved Recognition

of Subcellular Location Patterns in Fluorescence Microscope Images. Proc. SPIE,

4962, 307-318. (2003)

6 Mahalanobis, P. On the generalized distance in statistics. Proc. Natl. Inst. Sci. India

2, 49-55 (1936).

13Nature Chemical Biology: doi: 10.1038/nchembio.173