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METHODS
Cell culture and Reagents. Human umbilical vein endothelial cells (HUVEC) and
human pulmonary artery endothelial cells (HPAEC) were grown in Endothelial Growth
Medium-2-MV (EGM-2-MV) BulletKit (Clonetics, San Diego, CA). At 80-90%
confluence, the cells were serum-starved in medium containing EBM-2 and 0.5% fetal
bovine serum (FBS) for 16h and subsequently treated with thrombin at the doses and for
the times indicated.
Microarray analysis. HUVEC were serum-starved overnight (12h) in medium
containing EBM-2 and 0.5% FBS, then treated with 1.5 U/ml thrombin for varying
periods of time. RNA was harvested and purified with Trizol according to Manufacture’s
protocol (Invitrogen, Carlsbad, CA). Preparation of cRNA and hybridization of probe
arrays were performed according to the protocols of the manufacturer (Affimetrix, Santa
Clara, CA). Briefly, after quality determination on test arrays, the samples were
hybridized for 16h at 45oC to Affimetrix human focus array that have 8794 genes. The
arrays were washed and then stained with streptavidin-phycoerythrin. Fluorescently
stained probe arrays were visualized with GeneArray scanner (Hewlett-Packard). The
intensity for each feature of the array was captured with Affimetrix GeneChip software.
Pair wise comparisons were made by using probe arrays without thrombin treatment as
baseline. Duplicate samples from two independent batches of HUVEC (Clonetics, lot #
6541 and 2F0461) were analyzed for each of the three time points (1, 4 and 18h).
FileMaker software was used to examine the qualitative parameters of increase or
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decrease. Genes were identified as thrombin-responsive if they met the following criteria:
1) specific hybridization signals consistent with a ‘presence call’ derived from the 20
probe pairs representing each gene by means of a trimmed mean algorithm, and 2) at least
2-fold upregulation or downregulation in both experiments (compared with control
untreated). The data were clustered on the basis of similar regulation patterns using Gene
cluster (created by Dr. Eizen, Stanford University) and Java Tree View software (created
by Alok alok@genome) according to the manual on the default settings. Red and green
represent higher and lower expression than the median for that particular gene,
respectively. Color intensity is related to the difference with the median (black).
Ribonuclease (RNase) protection assays. HPAEC or HUVEC were grown in 6-well
plates, serum-starved overnight in EBM-2 medium containing 0.5% FBS and treated with
or without thrombin in the presence or absence of inhibitors, as described above. Cells
were harvested for RNA by adding RNA-STAT reagent (TEL-TEST Inc., Friendwood,
TX) directly into the wells. Total RNA was extracted according to the manufacturer’s
instructions. For RNase protection assays, a 440-bp TF, 376-bp PDGFA, 334-bp Egr-1,
283-bp ICAM-1, and 210-bp uPA human RT-PCR fragments were subcloned into the
pPCR-Script Amp SK(+) cloning vector using PCR-Script AMP Cloning Kit (Stratagene)
and were used as in-vitro transcription templates.. The cDNA inserts were verified by
automated sequencing. The cDNA plasmid template for -actin was purchased from
Ambion (Austin, TX). [-32P] UTP-labeled riboprobes were synthesized from the Egr-1
and -actin cDNA templates, using T3 RNA polymerase (Ambion) and subsequently gel
purified. RNase protection assays were carried out using the RPA IIITM kit (Ambion).
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Briefly, the two riboprobes were mixed with 5 µg total cellular RNA in a volume of 10 l
and hybridized at 42˚ C overnight in hybridization buffer. The unhybridized RNAs were
digested at 37˚ C for 30 min with RNase A /RNase T1 mix. The protected fragments
were precipitated by adding the RNase Inactivation/Precipitation III solution and
separated on a 4% acrylamide/8M urea gel. The gels were dried and autoradiographed.
Immunolocalization studies. HUVEC were plated onto glass cover-slides (Matsunami,
Japan) in the 6-well plate at a density of 25,000 cells/slide. The cells were grown in
EGM-2 MV medium for 24h and serum starved overnight in medium containing EBM-2
and 0.5% FBS, then treated in the presence or absence of 1U/ml thrombin for 1h, fixed in
ice-cold 3.7% paraformaldehyde for 10 min, washed with PBS and subsequently
incubated with primary anti-p65 antibody (Santa Cruz, Santa Cruz, CA). Following
extensive washes in PBS, the cells were incubated with a rhodamine-labeled secondary
antibody (1:200 dilution) (Santa Cruz) for 1h. The slides were washed in PBS, mounted
in Crystal/Mount (Biomeda, Foster City, CA) with Hoechst (Sigma) for identification of
nuclear localization and examined by fluorescence microscopy.
Western blot analyses. Western blot analyses were carried out as previously described 1.
The membranes were probed with anti-phospho Ser-256 FKHR (Cell Signaling, Beverly,
MA), stripped and reprobed with anti-FKHR to control for loading.
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LEGENDS TO ONLINE FIGURES
Figure I. Microarray analyses of thrombin treated endothelial cells. The Affymetrix
microarray (HG-FOCUS) expression data was clustered on the basis of similar regulation
patterns using Gene Tree view. Two independent batches of HUVEC were treated in the
absence (control) or presence of thrombin (1.5 U/ml) for 1, 4, 18h. The hierarchical tree
show the total cluster of all thrombin responsive genes after data filtering, including only
those genes that had ‘presence call’ and at least 2-fold upregulation (A) or 2-fold
downregulation (B) in both HUVEC samples compared to control.
Fig. II. Thrombin induces the nuclear translocation of p65 in primary endothelial
cells. HUVEC were serum-starved overnight in 0.5% FBS and incubated in the absence
(Control) or presence 1.5 U/ml thrombin for 1h. The cells were fixed in
paraformaldehyde, washed with PBS and subsequently incubated with primary anti-p65
antibody, followed by rhodamine conjugated second antibody. The nuclei were stained
with Hoechst.
Fig. III. Thrombin induces phosphorylation of FKHR in endothelial cells. Western
blot analyses of whole cell extract from HCAEC treated in the absence or presence of 1.5
U/ml thrombin for the times indicated were carried out using phospho-specific antibodies
to FKHR Ser-256. The membrane was reprobed for total FKHR. The result is
representative of 3 independent experiments.
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REFERENCES FOR ONLINE DATA SUPPLEMENT
1.
Abid MR, Tsai JC, Spokes KC, Deshpande SS, Irani K, Aird WC. Vascular
endothelial growth factor induces manganese-superoxide dismutase expression in
endothelial cells by a Rac1-regulated NADPH oxidase-dependent mechanism.
Faseb J. 2001;15:2548-2550.
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Fig. IA
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Fig. IB
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Fig. II
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Fig. III
Time (min)
0
5
15
p256-FKHR
FKHR
Thrombin
30