Virtakoivu et al 2015 – Supplemental Information

Supplementary figures 1-5 and associated legends

Supplementary materials and methods

Supplementary references

Figure S1. ERK silencing decreases breast cancer cell migration and invasion but does not

influence cell proliferation. (A) Migration of control-, ERK2-, vimentin- and Slug-silenced MDA-

MB-231 cells (top panel) was followed with time-lapse imaging for 13 h at 10 min intervals (n =

34-43 cells/condition). For invasion assays (bottom panel) control- and ERK2-silenced MDA-MB-

231 cells were allowed to invade in Matrigel for 4 days, stained with Alexa Fluor 488 phalloidin

and imaged with confocal microscopy. Invasion area was calculated from the side view (z-axis) of

invading cells and the arrow indicates the direction of cell invasion (n = 4). Silencing efficiencies

were analysed by western blot 72 hours post transfection. (B) Proliferation of control-, ERK-, Slug-

and vimentin-silenced MDA-MB-231 cells was analysed using WST-1 reagent (n = 2; 5-8 wells per

experiment). Silencing efficiencies were analysed by western blot 72 hours post transfection (mean

± SEM; * p < 0.05, *** p < 0.001). (C) Immunohistochemical staining of DAPI in orthotopically

implanted MDA-MB-231 primary tumors and representative contralateral lymph node metastasis

from three animals (n = 7 lymph node lesions and n = 5 primary tumors; these are from the same

samples as the vimentin staining shown in Fig 1). Scale bar: 300 µm and 100 µm for region of

interest (ROI) images.

Figure S2. ERK2 does not interact with actin or keratin 8 and ERK2 phosphorylation is

supported by vimentin but not actin. (A) Representative western blots of total ERK and pERK

levels in control or vimentin silenced MDA-MB-231 cells (n = 3). (B) ERK2 does not co-

immunoprecipitate (IP) with keratin 8 or actin. Inputs are shown for keratin 8 and actin protein

levels. (C) Quantification of ERK2 phosphorylation in alkaline phosphatase (AP) protection assays.

Recombinant active 32P-ATP phosphorylated ERK2 alone or in combination with vimentin, was

exposed to AP for the indicated times and protein phosphorylation detected by autoradiography (n =

4). (D) AP protection assay for active phosphorylated ERK2 in the presence of recombinant actin.

ERK2 alone or with actin was exposed to AP for the indicated times. Quantification of pERK levels

relative to total ERK are shown (n = 4). (E) Induction of stress fibers in MDA-MB-231 cells

transiently transfected with active RhoA mutants (GFP-RhoA QL or GFP-RhoA V14) or GFP

alone. Representative confocal images of actin stress fibres (phalloidin staining) and western blots

for active ERK (pERK), total ERK, actin and GFP are shown. (n = 3 experiments). Scale bar: 20

µm. (mean ± SEM; * p < 0.05).

Virtakoivu_Supplemental 3.

Figure S3. Fluorescence recovery of wild-type Slug-GFP after photobleaching (FRAP). (A-B)

Fluorescence intensities were measured from the indicated regions of interest (ROI) in the nucleus,

cytoplasm, and background and from another cell nucleus as a reference before and after

photobleaching. (A) A representative GFP-Slug signal in a cell at the indicated time points. (B)

Fluorescence intensity changes over time following photobleaching. Mono exponential fits were

used for recovery curves. (C) Quantification of the recovery half-time, mobile and immobile

fractions of GFP-Slug from 12 cells. The intensity values of analysed ROIs were corrected for

intensity changes of background and reference cells.

Figure S4. Mass spectrometric identification of ERK1/2-dependent phosphorylation sites on

Slug and validation using a newly generated Slug phospho-serine-87 antibody. (A) Slug was

preincubated with ERK1 or ERK2 in in vitro kinase assays. Following in-gel trypsin digestion and

TiO2 phosphopeptide enrichment, MS/MS analysis identified three phosphopeptides from ERK2-

phosphorylated Slug. (B) Label-free quantification of two of the identified phosphopeptides before

and after TiO2 phosphopeptide enrichment. Phosphopeptide abundance was normalized to Slug

protein abundance. (C) Specificity of Slug phospho-serine-87 antibody (anti-pSlug S87) was

analysed by measuring Slug phosphorylation levels in H-Ras transformed MCF10A-DCIS cells ±

MEK inhibitor (U0126, 24 or 48 h). Quantification of pSlug S87 levels from 3 independent

experiments is shown. (D) Antibody specificity was further validated in MCF10A cells expressing

Flag-Slug wt or Flag-Slug phospho-site mutants (S87A and S87,104AA constructs).

Figure S5. The phosphorylation status of Slug does not influence repression of E-Cadherin,

Slug nuclear localisation or stability. (A and B) E-cadherin promoter activity, measured with

Envision, in HEK293 and MCF7 cells co-transfected with luciferase construct for E-cadherin,

pIRES_slug wt/S87A/S87,104AA/dominant negative (DN) and pRL-TK plasmids (n = 3-5 for

HEK293 and n = 2 for MCF7 cells). (C) Western blot analysis of E-cadherin protein levels in

retrovirally-transduced MCF10A_GFP and MCF10A_GFP slug wt/mutant cells. (D) DNA binding

assays analysing GST-Slug wt or S87A/S104A mutant (Slug_AA) interaction with vimentin or E-

cadherin promoter DNA. Slug constructs were preincubated with active ERK2 and ATP in in vitro

kinase reaction buffer prior to performing the DNA binding assays (n = 3). (E) Slug localisation

was analysed by confocal microscopy in MCF10A cells transfected with GFP, GFP_Slug wt or

Slug phospho-site mutant. Cells were fixed and the localization of GFP_slug was quantified 24 h

post transfection (n < 90 cells/transfection). (F) Quantification of Slug localisation in fractionated

control or vimentin-silenced MDA-MB-231 cells (nuclear and cytosolic fractions) (n = 3). (G) Slug

protein stability was monitored in HEK293 cells transfected with pIRES_slug wt or pIRES_slug

S87,104AA plasmids. Cells were treated with cycloheximide (CHX) 10 µg/ml 24 h post

transfection, lysed at the indicated time points and subjected to western blot analysis. Quantification

of band intensities from six independent experiments analysed by ImageJ is shown (mean ± SEM; *

p < 0.05, *** p < 0.001).

Supplementary Materials and Methods

Antibodies, Reagents, siRNAs and Plasmids. The following antibodies were used in this study:

rabbit mAb antibodies against Slug (C19G7), p44/42 MAPK (Erk1/2) (137F5) and Phospho-p44/42

MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology) and 12G10 anti-alpha-tubulin

(Developmental Studies Hybridoma Bank), mouse monoclonal anti-vimentin, anti-Axl H-124, mouse

monoclonal IgG1 AXL (Z49M), mouse monoclonal lamin A/C IgG 2b (Santa Cruz Biotechnology),

goat anti-hAXL (R&D Systems), mouse monoclonal anti-beta-actin antibody (Sigma), rat

monoclonal anti-keratin-8 antibody (Troma I, XYZ, Developmental Studies, Hybridoma Bank, NIH,

USA), anti-integrin β1 MA2252 clone 29 (Millipore) and mAb GST antibody (GenScript). For

immunohistochemistry, primary polyclonal rabbit anti-human antibody for Slug (RB 1398) was

purchased from Abgent (San Diego, CA, USA) and for vimentin, clones V9 and HPA001762, from

Biogenex (Fremont, CA, USA) and Sigma, respectively. Antibodies directed against ERK and pERK

were from Cell Signaling.

Growth factors and/inhibitors used in this study included TGF-β1 (R&D systems) at 5 ng/mL

for 20 h (or for 5 days in TGFβ induction experiments), EGF (Sigma) at 50 ng/mL for 20 min and

MEK inhibitor U0126 (Sigma) at 10 µM for 20 h.

The following siRNAs were used in this study: ERK1 (MAPK3) siRNA (ON-TARGETplus

Smart pool Human MAPK3 siRNA LU-003592-00-0002: Dharmacon, Chicago, IL, USA), ERK2

(MAPK1) siRNAs (ON-TARGETplus Smart Pool Human MAPK1 siRNA LU-003555-00-0002,

Individual ON-TARGETplus MAPK1 siRNA J-003555-11-0002: Dharmacon), Slug siRNAs (ON-

TARGETplus Smart Pool siRNA L-017386-00-0005: Dharmacon, Hs_SNAI2_5 Flexitube siRNA

SI03034416: Qiagen), Vimentin siRNAs (Smart Pool M-003551-01-005: Dharmacon, #SI00302190:

Qiagen) and scrambled siRNA (AllStars negative control: Qiagen).

The following plasmids were used in this study: pcDNA3 wild type vimentin (full length),

pCMV_MEK_ERK2 plasmid, empty pCMV plasmid (used as a negative control), CRU5-IRES-GFP-

Slug, p3XFLAG-CMV-Slug and pEGFP-C1-Slug. The CRU5-IRES-GFP retroviral vectors for

expressing hSlug, shLuc and shAxl were conducted as described in (1). Point mutations in CRU5-

IRES-GFP-Slug, p3XFLAG-CMV-Slug and pEGFP-C1-Slug vectors were performed using

Quickchange II XL Site-directed mutagenesis kit according to the manufacturer’s instructions

(Agilent Technologies). SNAI1/Snail and SNAI2/Slug coding sequences were cloned into the pGEX-

4T-1 vector (Promega) and sequence verified. GST fusion proteins were expressed in Escherichia

coli (Rosetta BL21DE3) and purified according to the manufacturer´s instructions (BD Biosciences);

the GST moiety was cleaved with thrombin.

Cell Culture, Stable Cell Lines and Transfections. MDA-MB-231, MCF7 and HEK293 cells

were cultured in Dulbecco´s modified Eagle´s medium supplemented with 10 % FBS, 1 % L-

glutamate and non-essential amino acids (not included for MCF7 cells). MCF10A cells were

cultured in DMEM/F12 1:1 supplemented with 5 % horse serum, 20 ng/mL EGF, 0.5 μg/mL

hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin and 100 μg/mL streptomycin. Vimentin

WT and -/- mouse embryonic fibroblasts (MEFs) were immortalized by retroviral infection of

pBABE-Large T antigen (Biomedicum Genomics, Helsinki, Finland). Cells were split to 50%

confluence one day prior to the infections. Medium was changed the next day to include growth

medium (Dulbecco´s modified Eagle´s medium supplemented with 10 % FBS and 1 % L-

glutamate) and virus supernatant in 1:2 ratio. Cells were incubated with virus-containing medium

for 7h prior to media change, split 2 days after the virus infections and selected with 250 μg/ml

geneticin. Antibiotic selection was continued for 4 weeks.

siRNA transfections were performed using HiPerFect transfection reagent (Qiagen, Valencia, CA,

USA) according to the manufacturer´s protocol. Plasmid transfections were done using

Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer´s

protocol.

Tissue Preparation and Immunohistochemistry. Paraffin-embedded samples were cut into 4 µm

sections, mounted on SuperFrost Plus slides (Menzel-Gläser, Germany), deparaffinized in xylene,

rehydrated in graded alcohols, and rinsed in 0.01M phosphate-buffered saline (PBS). Microwave-

stimulated antigen retrieval was performed in 10 mM citrate buffer, at pH 6.0. An avidin-biotin-

peroxidase method was applied using Dako Envision Kit (Dakopatts, Copenhagen) or HistostainTM

Kit (Zymed Laboratories Inc). Endogenous peroxidase was blocked with aqueous 0.3% H2O2 for 15

min. Primary antibodies were diluted in PBS containing 1% bovine serum albumin (BSA) and 0.02

M glycine and PBS was used in all subsequent washing steps. Sections were incubated overnight

with primary antibodies directed against slug (dilution 1:100), vimentin (V9 clone, 1:3000), ERK

(1:100) and pERK (1:600). Diaminobenzidine (Sigma) was used as the chromogen and the sections

were lightly counterstained with Mayer’s hematoxylin. Samples were evaluated for the absence (-) or

presence (+) of nuclear or cytoplasmic immunoreactivity. The threshold for determining whether a

sample was positive for the expression of the indicated proteins was set at 20 % (i.e. percentage of

cells exhibiting positive IHC staining). Samples under this threshold were considered negative.

Immunohistochemical staining of frozen sections was carried out on Tissue-Tek® O.C.T.™

Compound (Sakura)-embedded sections. Following acetone fixation (10 min at -20oC) samples were

permeabilized in 0.3% Triton-X/2% BSA, blocked in 30 % horse serum-PBS (30 min) and stained

with anti-vimentin (1:400; HPA001762 clone) for 1 h at RT followed by PBS washes and incubation

with Alexa Fluor anti-rabbit-555 (1:400; Life technologies) and Dapi (0.1 ug/ml).

Tumor xenografts on chick embryo chorioallantoic membranes. Fertilized chicken eggs were

incubated like previously described in Hagedorn et al.2002. Shortly, to start the development the

eggs were washed and placed at 37 C incubator. After that on day 3, a small hole was made in the

eggshell in order to drop the chorioallantoic membrane (CAM). On the developmental day 10,

plastic ring was placed on CAM and 1 million control or vimentin silenced cells were implanted

inside the ring in 40 ul of 50 % matrigel. After 3 days tumors were imaged and dissected. Tumors

were lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% TX-100, 5% glycerol, 1%

SDS, 1 mM Na3VO4, 10 mM NaF, and 1 mM phenylmethanesulfonyl fluoride) and

homogenasized with MagNA lyser green beads in MagNA Lyser (Roche) (2).

Luciferase reporter assays and DNA binding ELISA. Dual-luciferase reporter assays were

performed according to manufacturer´s instructions (Promega, Madison, USA). Briefly, cells were

triple-transfected with pRL-TK, pE-cad-571-Luc (3) or pVIM-571-Luc (4, 5) and CRU5-IRES-GFP/-

Slug wt/-Slug mutant plasmids. Cells were lysed with Passive Lysis Buffer 48 hours post transfection

and plated on Costar 96-well plates (Corning, NY, USA). Luciferase Assay Substrate was added to

each well and luminescence measured with Envision multilabel plate reader (Perkin Elmer-Cetus,

Waltham, MA, USA). Stop & Glow reagent was added and the luminescence signal was re-

measured. The luciferase signals from E-cadherin and vimentin promoters were normalized for the

renilla luciferase signal (pRL-TK).

In DNA binding ELISA assays, 96-well plates (Corning, NY, USA) were incubated with

GST- Slug/SNAI2 WT or Slug S87A/S104A mutant for 1 hour at RT. Plates were blocked with

blocking buffer (5% BSA in TBS-T, 10 µM ZnCl2 and 1mM DTT) for 1 hour at RT and incubated

with DNA (300 ng/ml) in blocking buffer for 3 hours at RT. Plates were washed twice with blocking

buffer and incubated with SYBR® Gold (Molecular Probes) in 50 mM Tris pH 7.5. DNA was

detected by measuring sample emission at 520 nm using Envision 2100 multilabel reader (Perkin

Elmer).

qRT-PCR. Cellular RNA from transfected cells was isolated with Qiagen RNeasy kit and 20 ng of

the extracted RNA was used as a template for the reverse transcriptase reaction. 1/10 of synthesized

cDNA was then used for qRT-PCR. Primers for indicated proteins (designed using Roche´s

Universal ProbeLibrary) were used at 300 nM final concentration and probes at 100 nM. The

expression of indicated proteins was detected by the quantitation method using GAPDH as an

internal control.

Immunofluorescence, Fluorescence recovery after photobleaching and Flow Cytometry. For

immunofluorescence, cells were washed with cold PBS, fixed in cold 4% paraformaldehyde (PFAH),

permeabilized with 0.1% Triton X-100 in 2% BSA/PBS for 15 min at RT and blocked with 30 %

horse serum in PBS for 1 hour at RT. The indicated primary antibodies diluted in blocking buffer

were added and incubated for 1 h at RT. After washing three times with PBS, Alexa-conjugated

secondary antibodies and DAPI (4´6-diamidino-2-phenylindole) were added for 1 hour at RT.

Coverslips were washed with PBS and MQH2O and mounted with Vectashield mounting medium

(Vector Labs). Confocal 3D images were taken with Zeiss Axiovert 200 M with spinning disc

confocal unit Yokogawa CSU22 and Zeiss Plan-Neofluar 63×Oil/1.4 NA objective and analyzed with

NIH ImageJ.

For 3D immunofluorescence, MDA-MB-231 cells were embedded into 50% Matrigel and

allowed to form cell spheroids for 5 days. Cell invasion was then induced with 50 ng/ml EGF. 24 h

later, the cells were fixed and permeabilized simultaneously with 2% PFA in PBS supplemented with

0.5% Triton X-100 for 1.5 h at RT. After 3 consecutive washing steps (10-15 min per wash at RT)

with glycine buffer (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, and 100 mM glycine in

PBS), blocking was done for 2 h at RT with buffer containing 130 mM NaCl, 7 mM Na 2HPO4, 3.5

mM NaH2PO4, 7.7 mM NaN3, 0.1% BSA, 0.2% Triton-X100, 0.05% Tween20, and 10% horse

serum in PBS. Primary antibodies were used at 5–10 mg/ml concentrations in blocking buffer and

incubated overnight at 4 °C. The cells were then washed three times (for 20 min each step) with

blocking buffer at RT with gentle rocking. Alexa-conjugated secondary antibodies, at a concentration

of 5 μg/ml, were incubated at RT for 1 h, followed by another round of washing. The spheroids were

mounted with Mowiol containing anti-fading reagent (Vectashield, Vector Labs) for 1 h at 37°C.

Immunofluorescent samples were analysed with an inverted wide-field microscope (Carl Zeiss) with

a confocal unit, Orca-ER camera (Hamamatsu Photonics), Plan-Neofluar 63× oil/1.4 NA objective

(Carl Zeiss), and SlideBook 5.0 imaging software (Intelligent Imaging Innovations, Inc.).

For FRAP experiments MCF10A cells were plated on MatTek P35G-1.5-14-C glass-bottom

dishes (MatTek Corporation, Ashland, MA, U.S.A), and transfected 24 hours later with GFP-Slug wt.

FRAP measurements were performed 20 hours post transfection in Live-Cell Imaging Solution,

Molecular Probes (Life Technologies Corporation, NY, USA). Measurements were performed with

Carl Zeiss LSM780 confocal microscope equipped with an environmental chamber (set to 37 °C,

5.0% CO2), and using a 40x 1.2 W C-Apochromat objective (Carl Zeiss GmbH, Jena, Germany).

Slug-GFP was excited with the 488 nm laser (25mW Argon, LASOS Lasertechnik GmbH, Jena,

Germany), and emission was detected on Gasp-detector at a range of 490-570 nm. For each

measurement, images were scanned at 0.938 sec intervals using 0.60% of maximal excitation power

(25mW), and a pinhole size of 60 µm. Baseline intensities were collected during the first five frames,

followed by photobleaching of a full nuclear area to reduce its fluorescence intensity by 50-80% from

base line intensity. Recovery data was collected for 10-20 minutes. Another, reduced region of

interest (circle, diameter 5.30 µm) was chosen from the middle of nucleus for recovery analysis.

Analysis was done by using FRAP-module included in Carl Zeiss Zen 2010 software, version 6.0

(Carl Zeiss GmbH, Jena, Germany).

For FACS staining, cells were detached with HyQTase, fixed with 4% PFA for 15 min at RT,

washed with Tyrodes buffer (10 mM HEPES-NaOH at pH 7.5, 137 mM NaCl, 2.68 mM KCl, 1.7

mM MgCl2, 11.9 mM NaHCO3, 5 mM glucose, 0.1% BSA) and stained with primary antibodies

against Axl or with secondary antibody only in control cells for 1 h. Cells were then washed with

Tyrodes buffer and stained with Alexa Fluor 488-conjugated secondary antibody (1:400). After

washing, cells were suspended in Tyrodes buffer and fluorescence was analyzed by flow cytometry

(BD Accuri C6, BD Biosciences, USA). For STED-microscopy cells were plated on high-

performance cover slips, 170 ± 5 µm, No 1.5H (Marienfeld-Superior, Germany), fixed and stained as

described above. Vimentin was labelled with Mega 520-conjugated secondary antibody (1:100)

(Sigma-Aldrich, St. Louis, MO, USA), and Slug or pERK separately with Star 635-conjugated

secondary antibody (1:500) (Abberior GmbH, Göttingen, Germany).

Proliferation, Migration and Invasion. Proliferation, migration and invasion assays were

performed as described in (6) with some modifications. Briefly, inhibitor or siRNA-treated cells

([3–5] × 103) were plated on clear-bottomed Costar 96-well plates (Corning, Corning, NY). To

measure proliferation, 10 μl of WST-1 reagent was added to the cells 24 hours later, incubated for

45 min at 37ºC and absorbance was read at 450 nm with Envision multilabel plate reader (Perkin

Elmer-Cetus, Waltham, MA).

For analysis of cell migration, cells were allowed to adhere and spread overnight on culture

dishes prior to live-cell imaging. Phase-contrast images were taken every 10 min for 16–19 h with a

Zeiss inverted wide-field microscope (El Plan-Neofluar 10×/0.5 NA objective) equipped with a

heated chamber (37ºC) and CO2 controller (4.8%). Cell movement was tracked using the NIH

ImageJ software and ImageJ Plugin (MTrackJ).

Cell invasion was analysed on a 25% matrigel composition and performed as described in

(7), with some modifications; No inhibitory antibodies were included in this study and the siRNA

mix (70 nM siRNA and HiPerFect transfection reagent) was added to the Matrigel during the

polymerisation step.

Mass Spectrometry. To identify phosphorylation sites, following in vitro kinase assays proteins

were separated on SDS-PAGE gels and subjected to in-gel trypsin digestion, TiO2 affinity

chromatography and mass spectrometric analysis as described in (8) with some modifications. For

LC-MS/MS analysis, an EASY-nLC II nanoflow liquid chromatograph coupled to an LTQ Orbitrap

Velos mass spectrometer (Thermo Fisher Scientific) was used. Database search was performed

against the Swiss-Prot (Homo sapiens and E. coli) using Mascot 2.2 (Matrix Science) via Proteome

Discoverer 1.2 (Thermo Fisher Scientific). Label-free quantification was performed using Progenesis

LC-MS 3.0 (Nonlinear Dynamics).

In Situ Proximity Ligation Assay (PLA). This technique allows direct detection of protein-protein

interactions if the two proteins are in close proximity to each other (≈ 20 to 100 nm). The antigens of

interest must be recognized by antibodies raised in different species. Following antibody staining, the

cells are incubated with species-specific oligo-linked secondary antibodies. A PLA signal (which

appears as a bright fluorescent dot) is then produced by rolling circle amplification if the two

antibodies bind their antigens in close enough proximity and is indicative of protein-protein

interaction (9).

Briefly, MDA-MB-231 cells were plated on acid-washed coverslips, allowed to spread for 1

day and treated or not with 10 µM MEK inhibitor U0126, washed with PBS and fixed with 4%

paraformaldehyde for 10 min at RT. Cells were permeabilized with 0.1% Triton X-100 in 2%

BSA/PBS for 20 min and blocked with 2% BSA/PBS for 1 hour at RT. Parallel samples were

incubated as indicated with rabbit anti-pERK (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204))

and either mouse anti-vimentin or anti-GFP antibody (negative control) at 4 °C overnight. Proximity

ligation was performed according to the manufacturer’s instructions (Duolink in situ PLA, Olink

Bioscience). PLA signals were detected with Zeiss Axiovert 200 M with spinning disc confocal unit

Yokogawa CSU22 and Zeiss Plan-Neofluar 63×Oil/1.4 NA objective and analyzed with NIH ImageJ.

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