could play a significant role in the invasive process. It will be of great interest to define how FAs maintain a balance between adhesive and degradative properties. Materials and methods Antibodies and reagents MT1-MMP antibody (MAB3328) was purchased from Millipore. The anti- bodies against FAK, paxillin, and p130Cas were from BD. Anti-Src antibody (sc-18) and anti-GST (sc-138) were purchased from Santa Cruz Biotechnology, Inc. The phospho-Src family antibody pY416 was from Cell Signaling Technology, the monoclonal vinculin antibody was from Sigma-Aldrich, anti- Flag antibody from Cell Signaling Technology, and anti-GFP antibody from Roche. Goat anti–rabbit or goat anti–mouse secondary antibodies conjugated to either Alexa 488 or 594 were from Invitrogen. BB-94 was purchased from Tocris Bioscience. All the other chemicals and reagents, unless otherwise stated, were from Sigma-Aldrich. Cell culture Pancreatic cancer cell lines BxPC3, Panc 04.03 and PANC-1 cells were pur- chased from American Type Culture Collection (Manassas, VA). HT1080 cells were a gift from Dr. Zhenkun Lou (Mayo Clinic, Rochester, MN). BxPC-3 and Panc04.03 cells were maintained in RPMI plus 10% fetal bovine serum, and PANC-1 and HT1080 cells were maintained in DME plus 10% fetal bovine serum (Invitrogen), and all cell lines had 100 U/ml penicillin and 100 µg/ml streptomycin in 5% CO2 at 37°C. Cells were cultured in T-75 flasks (Thermo Fisher Scientific). Plasmid, siRNA, and transfection MT1-MMP-GFP and MT1-mCherry constructs were gifts from Drs. Roberto Buccione (Consorzio Mario Negri Sud, Chieti, Italy) and Philippe Chavrier (Institut Curie, Paris, France). FAK was amplified from a rat brain cDNA library and inserted into pEGFP-C1 or SFB vector (pIRES2-EGFP with S peptide, Flag tag, and streptavidin-binding peptide) as described pre- viously (Wang et al., 2011). p130Cas constructs were subcloned from pEBG-p130cas (Addgene). Constructs were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. siRNAs targeting MT1-MMP (D-004145-02), FAK (M-003146-02), and p130Cas (M-020465-01) were purchased from Thermo Fisher Scientific and trans- fected with Lipofectamine RNAiMAX (Invitrogen) following the standard protocol. Knockdown and reexpression experiments were performed as follows: two rounds of siRNA transfection in a 24-h interval, and 24 h after the second transfection, cells were rescued with indicated constructs. Immunoprecipitation and Western blotting Cells were lysed with NETN buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris, and 1 mM EDTA) at 4°C. Cell debris was removed by centrifugation, and the supernatant was incubated with 5 µg of the appropriate antibody and protein A beads at 4°C for 4 h. For the S protein IP, cell lysate was incu- bated with S protein Agarose (EMD) at 4°C for 2 h. The pellet was washed with NETN buffer three times, eluted in Laemmli sample buffer, and analyzed by Western blot as described previously (Wang et al., 2011). Fluorescence microscopy Cells were grown on 22-mm coverslips for transfections and immunocyto- chemistry. Cells were fixed in formaldehyde and processed as described previously (Wang et al., 2011). Images were taken with an AcioObserver microscope (Carl Zeiss) and Orca II camera (Hamamatsu Photonics), analyzed with iVision software (BioVision Technologies) and processed with Adobe Photoshop (Wang et al., 2011). Gelatin coating and degradation assay Acid-washed coverslips were first coated with 50 mg/ml poly-l -lysine for 20 min at room temperature, and then fixed with 0.5% glutaraldehyde for 15 min. Gelatin matrix was prepared by mixing 0.2% gelatin and Oregon Green gelatin (Invitrogen) at an 8:1 ratio. After coating for 10 min, coverslips were washed with PBS and quenched with 5 mg/ml sodium borohydride for 15 min followed by extensive wash. For degradation assay, cell suspen- sions were plated on top of these coated coverslips for designated amounts of time, and subjected to IF analysis as described above. Cells that de- graded ECM at FA sites were scored as positive, and more than 600 random selected cells were quantified (n > 200, three independent experiments). The percentage of cells with FA-mediated degradation was plotted and statistical significance was analyzed by two-sample t test. 384 JCB • VOLUME 196 • NUMBER 3 • 2012 Invasion assay This assay was performed in a Boyden chamber (#BW200L; Neuro Probe) with #PFA 8 filter (Neuro Probe). The filters were coated with 1 mg/ml growth factor–reduced Matrigel as described previously (Hauck et al., 2002). The lower chamber was filled with full serum medium, and cells suspended in serum-free medium were plated on the upper chamber. Cells were allowed to invade for 24 or 36 h at 37°C before fixing and IF analysis. Online supplemental material Fig. S1 shows ECM degradation at FAs in multiple cell lines. Fig. S2 shows that deficiency or inhibition of MT1-MMP abolishes ECM degradation at FAs. Fig. S3 shows that FA localization of p130Cas is FAK dependent, and that p130Cas knockdown does not affect FA integrity. Fig. S4 shows that phosphorylation on MT1 Y573 facilitates MT1–FAK interaction, as well as ECM degradation at FAs. Fig. S5 shows cell model–specific FA and invado- podia matrix degradation patterns. Online supplemental material is avail- able at http://www.jcb.org/cgi/content/full/jcb.201105153/DC1. Thanks to Robbin Eppinga and Kristi Simmons for reading the manuscript and helpful comments. Special thanks to Shaun Weller and Eugene Krueger for providing images as well as preparation of the manuscript. This study was supported by National Cancer Institute grant CA104125 to M.A. McNiven and the Optical Morphology Core of the Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK84567). The authors declare no conflicts of interest. Submitted: 26 May 2011 Accepted: 4 January 2012 References Artym, V.V., Y. Zhang, F. Seillier-Moiseiwitsch, K.M. Yamada, and S.C. Mueller. 2006. Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invado- podia formation and function. Cancer Res. 66:3034–3043. http://dx.doi .org/10.1158/0008-5472.CAN-05-2177 Bowden, E.T., E. Onikoyi, R. Slack, A. Myoui, T. Yoneda, K.M. Yamada, and S.C. Mueller. 2006. Co-localization of cortactin and phosphotyrosine identifies active invadopodia in human breast cancer cells. Exp. Cell Res. 312:1240–1253. http://dx.doi.org/10.1016/j.yexcr.2005.12.012 Buccione, R., G. Caldieri, and I. Ayala. 2009. Invadopodia: specialized tumor cell structures for the focal degradation of the extracellular matrix. Cancer Metastasis Rev. 28:137–149. http://dx.doi.org/10.1007/s10555- 008-9176-1 Caldieri, G., and R. Buccione. 2010. Aiming for invadopodia: organizing polarized delivery at sites of invasion. Trends Cell Biol. 20:64–70. http:// dx.doi.org/10.1016/j.tcb.2009.10.006 Caldieri, G., I. Ayala, F. Attanasio, and R. Buccione. 2009. Cell and molecular biology of invadopodia. Int Rev Cell Mol Biol. 275:1–34. http://dx.doi .org/10.1016/S1937-6448(09)75001-4 Chan, K.T., C.L. Cortesio, and A. Huttenlocher. 2009. FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast can- cer invasion. J. Cell Biol. 185:357–370. http://dx.doi.org/10.1083/jcb .200809110 Coopman, P.J., D.M. Thomas, K.R. Gehlsen, and S.C. Mueller. 1996. Integrin alpha 3 beta 1 participates in the phagocytosis of extracellular matrix molecules by human breast cancer cells. Mol. Biol. Cell. 7:1789–1804. Coopman, P.J., M.T. Do, E.W. Thompson, and S.C. Mueller. 1998. Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity. Clin. Cancer Res. 4:507–515. Courtneidge, S.A., E.F. Azucena, I. Pass, D.F. Seals, and L. Tesfay. 2005. The SRC substrate Tks5, podosomes (invadopodia), and cancer cell invasion. Cold Spring Harb. Symp. Quant. Biol. 70:167–171. http://dx.doi.org/10 .1101/sqb.2005.70.014 Cukierman, E., R. Pankov, D.R. Stevens, and K.M. Yamada. 2001. Taking cell- matrix adhesions to the third dimension. Science. 294:1708–1712. http:// dx.doi.org/10.1126/science.1064829 Cukierman, E., R. Pankov, and K.M. Yamada. 2002. Cell interactions with three- dimensional matrices. Curr. Opin. Cell Biol. 14:633–639. http://dx.doi .org/10.1016/S0955-0674(02)00364-2 Cunningham-Edmondson, A.C., and S.K. Hanks. 2009. p130Cas substrate domain signaling promotes migration, invasion, and survival of estrogen receptor- negative breast cancer cells. Breast Cancer (London). 2009:39–52. Davies, B., P.D. Brown, N. East, M.J. Crimmin, and F.R. Balkwill. 1993. A synthetic matrix metalloproteinase inhibitor decreases tumor burden and