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uPAR regulates the mitogenic activity of EGF
M. Jo et al.
SUPPLEMENTARY INFORMATION
“The Urokinase Receptor (uPAR) Primes Cells to Proliferate in Response to Epidermal
Growth Factor”
Minji Jo, Keena S. Thomas, Shinako Takimoto, Alban Gaultier, En-Hui Hsieh, Robin D.
Lester and Steven L. Gonias
Cloning of murine uPAR
Total RNA was isolated from uPAR+/+ MEFs using the RNeasy Mini kit (Qiagen). cDNA was synthesized using a (dT)17 primer and the Superscript First Strand
Synthesis kit (Invitrogen). The first 5’ RACE step was executed using a murine uPARspecific sense primer containing an EcoRI site and (dT)17 as the anti-sense primer. The
resulting products were amplified, in the second 5’ RACE step, using the same murine
uPAR-specific sense primer and a murine uPAR-specific anti-sense primer containing an
XbaI site. The resulting product was cloned into pCDNA 3.1 (Invitrogen) and the integrity of the construct was verified by sequenceing.
uPAR gene silencing
Human uPAR-specific duplex siRNA was synthesized by Dharmacon (Lafayette, CO).
The targeted sequence (gccgttacctcgaatgcat) corresponds to nucleotides 398-420 of the
coding region. The siRNA sequence was subjected to a Blast search to optimize specific
silencing. Pooled non-targeting siRNA was used as a control. siRNAs (0.1 M) were
introduced into MDA-MB 231 and MDA-MB 468 cells by incubation with Oligofectamine in serum-free medium for 4 h. The cultures were allowed to recover in serumcontaining medium for 24 h, according to the manufacturer’s instructions.
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uPAR regulates the mitogenic activity of EGF
M. Jo et al.
RT-PCR
Total RNA was isolated from MEFs using the RNeasy Mini kit. cDNA was synthesized
using the Superscript First Strand Synthesis kit and amplified with HotStar Taq polymerase master mix kit (Qaigen). The amplification products were separated by 1% agarose
gel electrophoresis and visualized by ethidium bromide staining. The primers for murine
uPA, murine EGFR and murine -actin were synthesized by Integrated DNA Technologies. (Coralville, IA).
Immunoprecipitation and immunoblot analysis
Cell extracts were prepared in ice-cold RIPA buffer (20 mM sodium phosphate, 150 mM
NaCl, pH 7.4, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid) containing protease inhibitor cocktail (Sigma-Aldrich) and sodium orthovanadate (1 mM). The protein concentration in each extract was determined by bicinchoninic acid assay (Sigma-Aldrich). To
detect the EGFR in MEFs, cell extracts were incubated with a slurry of concanavalin Aagarose (50%) (Vector Laboratories). Precipitates were subjected to SDS-PAGE on 8%
slabs, electrotransferred to nitrocellulose membranes, and probed with pan phosphoEGFR-specific antibody, phospho-Y845-specific antibody, or total EGFR-specific antibody. MEF extracts were analyzed directly, without affinity precipitation, to detect phosphorylated ERK, total ERK, and human uPAR. Cell extracts from breast cancer cells
were analyzed directly to detect all proteins.
To detect phosphorylated and total STAT5b, nuclear extracts were prepared from
MDA-MB 231 cells using NE-PER nuclear and cytoplasmic extraction reagents (Pierce).
The breast cancer cell nuclear extracts and whole cell extracts from MEFs were subjected
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uPAR regulates the mitogenic activity of EGF
M. Jo et al.
to immunoprecipitation using STAT5b-specific antibody. The immunoprecipitates were
washed three times with RIPA buffer and subjected to immunoblot analysis to detect
tyrosine-phosphorylated STAT5b. The breast cancer cell nuclear extracts were subjected
directly to immunoblot analysis to detect the nuclear marker, lamin, as a control for the
efficiency of the extraction procedure.
Cell growth and proliferation
Cell growth was measured by MTT assay, using the Cell Proliferation Kit I (Roche Molecular Biochemicals). MEFs and MDA-MB 231 cells were cultured in serum-free medium for 4 h. uPA, EGF, or vehicle was added for 48 h. MTT hydrolysis was determined
by the absorbance at 570 nm. Control cultures were analyzed prior to the 48 h incubation
with uPA or EGF. The percent change in cell number during the 48 h culture period is
reported. Each result represents the average of three different experiments, each with
eight internal replicates (mean±SEM, n=3).
Cell proliferation was measured by BrdU incorporation, as previously described. MEF and MDA-MB 231 cells were plated on coverslips in 12-well plates. Cells
were cultured in serum-free medium for 18 h and then treated with EGF or vehicle and
BrdU (100 M). The incubation time with BrdU was 4 h with MEFs and 18 h with
MDA-MB 231 cells. Cells were then incubated with BrdU-specific mouse antibodyAlexa Fluor 594 conjugate (1:50) for 18 h. Incorporation was detected by counting the
fraction of immunofluorescent cells. When cells were treated with antibodies (25 g/mL),
the cells were first subjected to a mild acid wash prior to adding EGF and BrdU.
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uPAR regulates the mitogenic activity of EGF
M. Jo et al.
To selectively detect BrdU incorporation in transiently transfected cells, the expression construct of interest or empty vector and pEGFP were co-transfected at a ratio of
1:4. The cells were fixed with 4% formaldehyde, treated with 2 M HCl for 1 h, and then
incubated with BrdU-specific antibody-Alexa Fluor 594 conjugate together with GFPspecific polyclonal antibody-Alexa Fluor 488 conjugate (1:200). These cells were then
incubated with rabbit-IgG-specific secondary antibody-Alexa Fluor 488 conjugate
(1:200) for an additional 1 h assuring intense green fluorescence of the cells that were
transfected with pEGFP. The fraction of transfected cells, which were also red-fluorescent, was determined. At least four fields of three different coverslips were reviewed in
each experiment. All results are expressed as the fold-increase in BrdU incorporation
relative to that observed in cells treated with vehicle. All experiments were performed in
triplicate. Statistical significance was determined by unpaired t-test.
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