invasion and motility

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1 Supplementary Data Maria José Oliveira, Jozef Van Damme, Tineke Lauwaet, Veerle De Corte, Georges De Bruyne, Gerda Verschraegen, Mario Vaneechoutte, Marc Goethals, Mohammad Reza Ahmadian, Oliver Müller, Joël Vandekerckhove, Marc Mareel, and Ancy Leroy β-casein-derived peptides, produced by bacteria, stimulate cancer cell invasion and motility

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4 Section Figure Legends Fig. 1. The pro-invasive 13-mer peptide promotes cellular migration and motility. (A) HCT- 8/E11 cells were grown until confluency on top of glass coverslips, in a 6-well-plate. Confluent monolayers were wounded and incubated, in serum-free medium, with or without the pro-invasive 13-mer peptide. At fixed places (6/wounded monolayer), the width of the wound at time 0, 2, 4, 6, 10, 24 and 48 h was measured. Migration is expressed as the average ± SD (flags) of the difference between the initial width of the wound (at time 0) and the width registered after 2, 4, 6, 10, 24 or 48 h. *, Significantly different from untreated monolayer at P<0.01. (B) The pro-invasive 13-mer peptide promotes motility of cells located at the front of a wounded monolayer. Phalloidin-FITC staining of wounded monolayers treated with (+) or without (-) the 13-mer peptide for 6 or 10 h. Scale bar represents 20 µm. Fig. 2. Naloxone, an δor antagonist, blocks 13-mer peptide-mediated invasion. HCT-8/E11 cells were incubated, on top of collagen type I gels for 24 h, with the 13-mer peptide (10-7 M) and with naloxone (0, 10-10, 10-9, 10-8, 10-7, 10-6 or 10-5 M). Invasion indices (%) were determined as described in Material and methods. Bars represent mean values of, at least, three independent experiments and flags indicate standard deviations. *, Significantly different from untreated cells at P<0.005.

5 Section Materials and methods RP-HPLC-MS and RP-HPLC-MS/MS analysis CM were partially purified by HPLC on a C-18 reverse phase narrow-bore column (Vydac, Hesperia, CA). Part of the flow (5%) was directed into the electrospray ion-source of a Q- TOF mass spectrometer (Waters, Milford, MA) to perform on-line RP-HPLC-MS. The remaining flow (95%) was manually collected in fractions, lyophilised, dissolved in serumfree medium, and used for functional analysis. In pro-invasive fractions, differential ions, present in CM Coll Listeria/TSB and absent in CM Coll TSB, were selected for RP-HPLC- MS/MS. Peptide identification was achieved by submitting RP-HPLC-MS/MS data to Mascot ( and by presenting de novo deduced sequences to BLAST ( databases. Peptide synthesis The 33-mer peptide was synthesized by Fmoc. chemistry, using a 413A Peptide Synthesizer (Applied Biosystems, Foster City, CA). Other β-casein peptides were synthesized by tboc. chemistry. Synthetic peptides were purified by RP-HPLC (>97% purity), amino acid composition determined by amino acid analysis and amino acid sequences confirmed by RP- HPLC-MS/MS. Transfections HCT-8/E11 cells were transiently transfected with LipofectAMINE (Invitrogen). Twenty-four hours after, cells were used for collagen invasion assay and Immunoblot analysis. Equal loading was confirmed by immunoblotting with anti-α-tubulin (Sigma). cdna constructs for dominant-negative RhoA (RhoAN19), Cdc42 (Cdc42N17) or Rac1 (Rac1N17) or

6 constitutively active RhoA (RhoAL63), Cdc42 (Cdc42L61) or Rac1 (Rac1L61) expression were kindly provided by Dr. A. Hall (University College London, London, UK). The cdna construct for the PAK1 kinase dead mutant pcmv6m-hpak1k299r (PAK1K299R) was kindly provided by Dr. J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA). The cdna construct for p85 ish2n was a kind gift of Dr. B. Vanhaesebroeck (Ludwig Institute for Cancer Research, London, UK). Immunoblot analysis Cells were lysed in cold lysis buffer (20 mm Tris-HCl ph 7.5, 150 mm NaCl, 1% Triton X- 100, 1% NP-40, 3 mm sodium vanadate, 20 mm NaF, 1 mm PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin). Proteins (30 µg) were separated by SDS-PAGE under reducing conditions and transferred onto Hybond nitrocellulose membranes (Amersham, Buckinghamshire, UK). After blocking with 4% bovine serum albumin (Sigma) in PBS+0.5% Tween-20, membranes were incubated for 1 h with mouse monoclonal antibodies directed against Rac1, myc (Upstate Biotechnology), RhoA (Santa Cruz Biotechnology, Santa Cruz, CA), FAK (Transduction Laboratories, San Diego, CA), or v-src (Oncogene, Darmstadt, Germany), with rabbit polyclonal antibodies directed against Cdc42, δor, PAK1 (Santa Cruz Biotechnology), p85 subunit of PI3K (Transduction Laboratories) or with the horseradish peroxidase (HRP)-conjugated recombinant anti-phosphotyrosine antibody (RC20-HRP, Transduction Laboratories). Sheep anti-mouse or donkey anti-rabbit HRP-conjugated secondary antibodies (Amersham) were used, followed by ECL detection (Amersham). Immunoprecipitation After treatment with β-casein synthetic peptides, cells were lysed in cold lysis buffer. Sevenhundred micrograms of proteins were incubated for 2 h at 4ºC with a mouse monoclonal anti-

7 phosphotyrosine antibody (PY-20) (Transduction Laboratories), an anti-v-src antibody or with an anti-phosphoserine antibody (Sigma). Immunocomplexes were incubated for 1 h with protein G-sepharose beads (Amersham), centrifuged, washed three times and boiled for 5 min in sample buffer. Proteins were separated by SDS-PAGE and visualized by Immunoblot analysis. Wound healing assay HCT-8/11 cells were grown till confluency in 6-well tissue culture plate (Nunc). Monolayers were incubated for 15 minutes in Moscona s buffer and a scar was made with a 10 µl plastic pipette tip. Wounded monolayers were washed twice with PBS, to remove floating cells, and subsequently treated or not with the 13-mer peptide in serum-free medium. At the underside of each well, six reference marks were made. At each of these places, the width of the wound at time 0, 2, 4, 6, 10, 24 and 48 h was measured. Migration is expressed as the average ± SD of the difference between the initial width of the wound (at time 0) and the width registered after 2, 4, 6, 10, 24 or 48 h. At each time point, wounded monolayers, growing on glass coverslips, were fixed for 20 min in 3% paraformaldehyde for FITC-phalloidin staining.