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Sigma-Aldrich, Zwijndrecht, The Netherlands). The cells were selected by puromycin (sc-108071, Santa
Cruz Biotechnology, Heidelberg, Germany). Knockdown efficiency was verified by Western Blot and
immunofluorescent staining.

In vivo tumor growth and metastasis formation
Six-week-old Rag2 gc mice were obtained from in-house breeding. Animals were housed in individually
-/-
-/-
ventilated cages under sterile conditions containing 3 mice per cage. Sterilised food and water were provided
ad libitum. To measure spontaneous metastasis, tumor cells (5 × 10 ) were injected into the right thoracic
5
mammary fat pads as described previously . After 4 weeks, the lungs were excised as well as the primary
[10]
tumor which was also weighed. For quantification of all GFP-paxillin positive macro- and micro-metastases,
the flat side of the right lung was analysed with the immunofluorescence microscope using a 10× objective
lens (NA 0.25). Next the right lung and primary tumor were fixated in 4% paraformaldehyde. Paraffin
sections of the lungs (5 mm) were stained with hematoxylin and eosin followed by histological analysis.

Statistical analysis
When not indicated, all experiments were performed in biological triplicates. As all data sets in this study
follow a normal distribution, therefore were compared with Student’s t-test (two-tailed, equal variances)
using GraphPad Prism 6.0. Results were considered to be significant if P value < 0.05.

RESULTS
EGF-induced cell migration of MTLn3 cells is dependent on JNK activation and associated with
paxillin Ser178 phosphorylation
First we determined the role of JNK-paxillin signaling in EGF-induced migration of the highly metastatic
MTLn3 rat mammary adenocarcinoma cell line. EGF exposure caused membrane ruffling [Supplementary
Figure 1], random cell migration and scattering of MTLn3 cells [Figure 1A and Video 1]. An inhibitor
of JNK, SP600125, blocked the migration almost completely, which was associated with increased cell
clustering of the MTLn3 cells. This clustered phenotype was associated with increased localization of
β-catenin at cell-cell contacts [Figure 1B and Supplementary Figure 2]. Visual inspection of the movies
indicates that inhibition of JNK reduces persistent movement [Figure 1C] and quantitative analysis of the
cellular tracks reveal that the velocity of both individual and clustered cells was significantly impaired
[Figure 1D]. By immunofluorescence, we observed that active phosphorylated JNK co-localizes with paxillin
at focal adhesions in MTLn3 cells [Supplementary Figure 3B] as was previously observed in renal epithelial
cells [47,48] . EGF treatment induced a transient phosphorylation of paxillin at Ser178 in association with JNK
activation, while SP600125 prevented phosphorylation of JNK, c-Jun as well as paxillin at Ser178 indicating
the importance of EGF-induced JNK activation for paxillin phosphorylation [Figure 1E and Supplementary
Figure 3C]. Together, these data indicate that in MTLn3 cells EGF-induced JNK activation mediates cell
migration possibly via the phosphorylation of paxillin at Ser178.

PaxillinS178A mutant inhibits MTLn3 cell motility in vitro
To further investigate the role of paxillin Ser178 in cell migration, we generated MTLn3 cell-lines stably
expressing either GFP-tagged paxillin-wt or phospho-defective mutant GFP-paxillin in which the serine
residue 178 was replaced by alanine (further referred to as paxillinS178A). Three independently obtained
paxillin-wt clones and paxillinS178A clones were selected for further experiments. MTLn3 clones stably
expressing GFP-wt-paxillin or GFP-paxillinS178A were evaluated by flow cytometry, Western blotting
[Supplementary Figure 4A] and immunofluorescence [Figure 2A]. Expression levels were approximately
equal in all clones. Although GFP-paxillinS178A still localized at focal adhesions, cells were smaller and
clustered with enhanced β-catenin-containing cell-cell contacts while cells expressing the GFP-wt-paxillin
were stretched with large lamellipodia and almost no cell-cell contacts [Figure 2A and Supplementary
Figure 4B]. The paxillinS178A-induced cell clusters [Figure 2B and Supplementary Figure 4C] resembled