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John P. Hobson et al. Supplementary Methods Enzymes for DNA manipulation and modification were purchased from New England Biolabs (Beverly, MA). PrAg, PrAg-U2, PrAg-U7, PrAg-L1, and PrAg-33 were generated and purified as described previouslys1-3. Reagents purchased from American Diagnostica (Stamford, CT) included pro-uPA, human uPA amino terminal fragment (ATF), active plasminogen activator inhibitor (PAI)-1, and human Glu-plasminogen (Plg). BB-94, BB-2516, and tissue inhibitor of metalloproteinase-2 (TIMP-2) were obtained from Vernalis (Cambridge, UK). Nitrocefin was purchased from Oxoid (Hampshire, England). Coumarin cephalosporin fluorescein acetoxymethyl ester (CCF2/AM) and loading solutions were purchased from Invitrogen (Carlsbad, CA). Antitrypsin Portland was purchased from Affinity BioReagents (Golden, CO). Cells and tissue culture. HeLa and HT1080 cells were obtained from American Type Culture Collection (Manassas, VA). Wild type Chinese hamster ovary (CHO) cells and furin-deficient CHO cells (FD-11), wild-type and isogenic uPA receptor (uPAR)-deficient mouse dermal fibroblasts, HT-1080 fibrosarcoma cells, HN6, HN12, HN13, HN26, and HN30 head and neck squamous cell carcinoma cell lines have been describeds1, 4-10 . All cells were maintained in phenol red-free DMEM (Gibco-BRL, Gaithersburg, MD) supplemented with 10 % fetal bovine serum (FBS), glutamine, and gentamicin reagent solution. 125I -LF binding and internalization. LF was labeled with 125I using Iodogen-coated tubes from Pierce (Rockford, IL). Briefly, 20 g of purified LF was incubated with 0.1 M Tris-HCl (pH 7.6) with 1 mCi 125 I to make a final reaction volume of 100 l. The reaction was incubated for 10 min at room 1 John P. Hobson et al. temperature and terminated with 900 l of 0.1 M Tris-HCl (pH 8.1) with 0.01% Tween80, followed by gel filtration using Sephadex G25 column PD10 from Pharmacia Biotech (Arlington Heights, IL). Aliquots were then subjected to analysis by SDS-PAGE and quantitation by -counting to determine the specific activity of the labeled protein. Binding and internalization of 125 I-LF was done using CHO cells that were seeded onto 24-well plates and cultured to near confluence. Briefly, the cells were washed twice in binding buffer (DMEM supplemented with glutamine and 4 mg/ml BSA) and incubated with 13 nM (1 g/ml) wild type PrAg or PrAg-U7 in the presence of 11 nM (1 g/ml) 125 I-LF at 37 °C for 1 h. The cells were then washed twice with ice-cold PBS, and incubated for 2 min at 4 °C with 50 µg/ml trypsin and 0.5 mM EDTA in PBS to strip non-internalized 125I-LF from the cells. The detached cells were centrifuged at 3,000 rpm for 5 min at 4 °C, and the radioactivity in the pellet (internalized LF) and supernatant (cell surface-bound LF) were measured with a radiation counter. Construction and purification of LF254-lactamase (LF/-Lac) fusion protein. The LF/-Lac fusion protein was generated by fusing the PrAg-binding region of LF (amino acids 1-254) N-terminal to the TEM-1 -lactamase gene from pBluescript (Stratagene, La Jolla, CA). lactamase were PCR A DNA fragment encoding amino acids 19-286 of - amplified from pBluescript using the primers 5’- GTAGGTCGACAAGGTGGTCTTCCTGTTTTTGCTCACCCAGAAACGC-3’ and 5’AGATGTCGACTTATTACCAATGCTTAATCAGTGAGGC-3’ furnished with SalI restriction sites for cloning. The resulting PCR product was digested with SalI, gel purified, and cloned into the LF254 fusion vector pGEX-KG-LFs11, which also furnished the protein with a GST tag and a thrombin cleavage site to facilitate purification. The 2 John P. Hobson et al. expression plasmid was then transformed into BL21 competent bacteria (Invitrogen, Carlsbad, CA), and grown in Super Broth (Biosource, Camarillo, CA) supplemented with 50 g/ml ampicillin. For protein purification, cultures were grown at 37 °C until an absorbance of 0.8 at 600 nm was reached. The cells were then induced with isopropyl D-thiogalactopyranoside at a final concentration of 0.5 mM for 5 h at 37 °C. The cells were pelleted and resuspended in bacterial lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1 % Triton X-100, and protease inhibitors. Bacterial pellets were then lysed by five rounds of freeze/thawing, followed by sonication for 30 seconds. The lysate was clarified by centrifugation and incubated with glutathione Sepharose resin from Amersham Biosciences (Piscataway, NJ) for 2 h at 4 °C. After incubation, the resin was washed twice with lysis buffer without protease inhibitors, and the purified protein was released from the resin by incubation with thrombin. An aliquot of the resulting sample was then analyzed by SDS-PAGE followed by staining with Coomassie brilliant blue to visualize purity. The enzymatic activity of the purified LF/-Lac fusion protein was verified using Nitrocefin colorimetric substrate, as per manufacturer’s protocol. Protease activity imaging assays. Fluorescent microscopy analysis. For protease activity imaging by fluorescence microcopy, cells were seeded at a density of 2x105 cells/well on eight-well chamber slides that were treated with poly-D-lysine. The cells were washed twice (except head and neck cancer monocultures) in DMEM and incubated with 26 nM (2 g/ml) PrAg or reengineered PrAg and 90 nM (5 g/ml LF/-Lac) fusion protein for 1 h at 37 °C. After incubation, the cells were washed twice with DMEM and loaded with 1.5 M CCF2/AM using the alternative substrate loading solution method from Invitrogen, except for mouse 3 John P. Hobson et al. fibroblast cultures, which were loaded using the general loading protocol. The cells were loaded for 1 h at room temperature, washed three times with DMEM, and incubated for additional 1 h at room temperature to allow fluorescence resonance energy transfer (FRET) disruption. Samples were visualized and photographed using a Zeiss Axioplan inverted microscope with Zeiss Axiovision software (Carl Zeiss, Jena, Germany). For acquisition of blue fluorescence, excitation filter HQ405/20 nm bandpass, dichroic 425DCXR, and emitter filter HQ460/40 nm bandpass were used. For green fluorescence, HQ405/20 nm bandpass, dichroic 425DCXR, and emitter filter HQ530/30 nm bandpass were used. All filters and dichroic mirrors were purchased from Chroma Technology (Rockingham, VT). All images were acquired using the identical settings. Statistical analysis was performed with Student’s t-test, two-tailed. Quantitative blue and green fluorescence measurements were performed in parallel using a using a VICTOR plate reader (see below). Blue-green fluorescence ratio determinations were calculated as described by Invitrogen, using octuplicate determinations for PrAg-33, triplicate determinations for PrAg-U2, and quadruplicate determinations for PrAg-L1. Microtiter plate assay. Cells were seeded 24 h before the assay at a density of 2x104 cells/well on poly-d-lysine treated 96-well black wall plates with a clear bottom. The cells were washed twice in DMEM and incubated with 26 nM (2 g/l) PrAg or reengineered PrAg mutants and 90 nM (5 g/ml) LF/-Lac fusion protein for 1 h at 37 °C. After incubation, the cells were washed twice with DMEM and loaded with 1.5 M CCF2/AM using the alternative substrate loading solution method from Invitrogen as described above. Data were collected using a VICTOR plate reader (Perkin Elmer, Wellesley, MA) equipped with the following filters in dual read mode: excitation 405/10 4 John P. Hobson et al. nm bandpass, emission filters 460/25 nm bandpass for blue fluorescence and 535/25 nm bandpass for green fluorescence. Flow Cytometry. Flow cytometry experiments were carried out using wild type and furin-deficient CHO cells grown on 10 cm tissue culture dishes. The cells were washed twice with DMEM and incubated with 26 nM (2 g/ml) PrAg or PrAg-U7 and 90 nM (5 g/ml) LF/-Lac for 1 h at 37 °C. After incubation, cells were washed twice with DMEM and loaded with CCF2/AM for 1 h using the alternative substrate loading solution method from Invitrogen. The cells were washed three times with DMEM and incubated for additional 1 h at room temperature to allow FRET disruption. After the final incubation, the cells were removed from the plate using trypsin-EDTA solution, washed twice in ice-cold Hank’s buffered salt solution containing 2 mM probenicid (Sigma, St. Louis, MO) and resuspended at a concentration of 106 cells/ml. The cells were analyzed on a BD LSRII flow cytometer (Beckton Dickenson San Jose, CA) equipped with a standard 488 nm laser and a 405 nm violet laser. The BD LSRII was also equipped with the following filter set and dichroic mirror: 525/50 nm for green light, 440/40 nm for blue light, and a 505 LP dichroic mirror. Ratiometric data analysis was done using Flow-Jo analysis software (TreeStar Inc., Ashland, OR) as described previouslys12. References 1. 2. Gordon, V.M., Klimpel, K.R., Arora, N., Henderson, M.A. & Leppla, S.H. Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases. Infect Immun 63, 82-87. (1995). Liu, S., Bugge, T.H. & Leppla, S.H. Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin. J Biol Chem 276, 17976-17984. (2001). 5 John P. Hobson et al. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Liu, S., Netzel-Arnett, S., Birkedal-Hansen, H. & Leppla, S.H. Tumor cellselective cytotoxicity of matrix metalloproteinase-activated anthrax toxin. Cancer Res 60, 6061-6067 (2000). Burns, J.E. et al. The p53 status of cultured human premalignant oral keratinocytes. Br J Cancer 70, 591-595 (1994). Cardinali, M., Pietraszkiewicz, H., Ensley, J.F. & Robbins, K.C. Tyrosine phosphorylation as a marker for aberrantly regulated growth-promoting pathways in cell lines derived from head and neck malignancies. Int J Cancer 61, 98-103 (1995). Levy, A., Hall, L., Yeudall, W.A. & Lightman, S.L. p53 gene mutations in pituitary adenomas: rare events. Clin Endocrinol (Oxf) 41, 809-814 (1994). Prime, S.S. et al. Epidermal growth factor and transforming growth factor alpha characteristics of human oral carcinoma cell lines. Br J Cancer 69, 8-15 (1994). Prime, S.S. et al. TGF-beta receptor regulation mediates the response to exogenous ligand but is independent of the degree of cellular differentiation in human oral keratinocytes. Int J Cancer 56, 406-412 (1994). Yeudall, W.A., Crawford, R.Y., Ensley, J.F. & Robbins, K.C. MTS1/CDK4I is altered in cell lines derived from primary and metastatic oral squamous cell carcinoma. Carcinogenesis 15, 2683-2686 (1994). Yeudall, W.A. et al. Ras gene point mutation is a rare event in premalignant tissues and malignant cells and tissues from oral mucosal lesions. Eur J Cancer B Oral Oncol 29B, 63-67 (1993). Arora, N. & Leppla, S.H. Fusions of anthrax toxin lethal factor with shiga toxin and diphtheria toxin enzymatic domains are toxic to mammalian cells. Infect Immun 62, 4955-4961. (1994). Knapp, T., Hare, E., Feng, L., Zlokarnik, G. & Negulescu, P. Detection of betalactamase reporter gene expression by flow cytometry. Cytometry A 51, 68-78 (2003). 6