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Verkoeijen et al. J Cancer Metastasis Treat 2019;5:51 I http://dx.doi.org/10.20517/2394-4722.2019.06 Page 3 of 16

paxillin by JNK can regulate cell migration and metastasis formation via modulation of the EGFR-signaling
pathway.

METHODS
Chemicals and antibodies
Alpha modified minimal essential medium without ribonucleosides and deoxyribonucleosides (a-MEM),
fetal bovine serum (FBS), phosphate-buffered saline (PBS), trypsin and geneticin (G418 sulphate) were from
Life Technologies. Rat tail collagen type I was from Upstate Biotechnology. LipofectAMINE Plus transfection
reagents were from Invitrogen. Primary antibodies were anti-paxillin (BD), anti-tubulin, anti-GFP (Sigma,
St. Louis, MO), anti-pT183/pY185-JNK (Promega), anti-Phospho-Thr202/Tyr204 ERK1/2, anti-pSer473-
AKT (Cell Signaling), anti-pSer178-paxillin (Abcam, Cambridge, UK), anti-EGFR for Western blot (rabbit
polyclonal, Cell Signaling Technology) and FACS/immunostaining (mouse monoclonal, Calbiochem). All
secondary antibodies were from Jackson. The Western-Star immunodetection system (Tropix kit) was from
Applied Biosystems and ECL Plus reagent was from Amersham. Hoechst 33258 and rhodamine-phalloidin
were from Molecular Probes and Aqua Poly/Mount was from Polysciences.

Cell culture
MTLn3 cells were cultured as before . To generate stable cell lines, MTLn3 cells were transfected with GFP-
[42]
paxillin or GFP-paxillinS178A kindly provided by Huang et al. were transfected using LipofectAMINE
[24]
plus reagents according to manufacturer’s procedures. Stable transfectants were selected using G418 and
individual clones were picked and kept at 100 mg/mL G418. For EGF experiments, 70%-80% confluent
cells were starved for 4 h followed by exposure to EGF (10 nmol/L). Human EGFR was expressed in GFP-
paxillin S178A using retroviral transfection followed by FACS sorting as described previously . Retroviral
[43]
transduction of GFP-paxillin cells with a pMSCV-blast-hEGFR retroviral vector, followed by blasticidin
selection (12.5 mg/mL) was used to generate GFP-paxillin S178A EGFR cells.

Luciferase reporter assay
MTLn3 cells were transiently transfected with the reporter constructs pGL3-Tata-5xE3AP1 , pGL3-Tata-
[44]
5xCol1TRE, pGL3-Tata-5xJun2 kindly provided by Dr. Hans van Dam (LUMC) using Lipofectamine Plus
reagent . 1 µg of expression reporter plasmids together with 1 µg of a renilla-luciferase construct (as an
[42]
internal control for transfection efficiency) were transfected in both clones WT and S178A. After 48 h, cells
were serum starved for 4 h then lysed and analyzed for luciferase activity using a luminescence plate reader.

Proliferation, attachment and wound healing assay
For proliferation assay, cells were plated in complete medium in 6 wells-plates and cultured for 24, 48, 72
or 96 h at which time point the amount of cells was determined by counting. For cell attachment assay, 1 h
serum-starved cells were seeded in complete medium on collagen-coated 6 wells-plates for 30, 60, 90 and
120 min, and following a PBS wash, attached cells were trypsinized and counted. For wound healing assays,
monolayer cells were scratched using a pipette tip to generate a wound followed by a wash with medium and
incubation in a-MEM supplemented with 1% (v/v) FBS for 20 hrs. Wounds were photographed using phase
contrast microscopy with a Nikon Coolpix digital camera directly after scratching and after 20 h. Wound
closure was determined using Image J software.

Live cell imaging
Random cell migration
Cells were cultured in glass-bottom plates overnight and starved for 4 h followed by imaging for 1-3 h
on a Nikon TE 2000-E microscope in a humid climate of 37 °C and 5% CO2 with either DIC (Differential
Interference Contrast) or fluorescence microscopy. Subsequently, cells were treated with EGF and time-