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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
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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
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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
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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
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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.
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