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SUPPLEMENT MATERIAL
Materials and Methods
Materials
Minimum essential medium, penicillin, SDS, Triton X-100, agarose, sodium acetate,
trypan blue, L-arginine, urea, and dialyzed serum were from Sigma-Aldrich (St. Louis, MO).
Antibodies against arginase I, cyclin D1, cyclin E, cyclin A, p21, p27, and β-actin were from
Santa Cruz Biotechnology (Santa Cruz, CA). S-(2-boronoethyl)-L -cysteine (BEC), NG-hydroxynor-L-arginine (L-OHNA), and a p53 antibody were from EMD Biotechnology (San Diego,
CA). α-[32P]dCTP (3000 Ci/mmol) and [guanido-14C]L-arginine (52 Ci/mmol) were from
Amersham Life Sciences (Arlington Heights, IL). Rat arginase I siRNA (Smartpool) and nontargeting siRNA was purchased from Dharmacon Inc (Lafayette, CO).
Cell Culture
VSMCs were isolated by elastase and collagenase digestion of rat thoracic aorta and
characterized by morphological and immunologic criteria. Cells were serially cultured in
minimum essential medium containing 10% dialyzed serum, Earle’s salts, 5.5 mM glucose, 2mM
L-glutamine, 5mM Tes, 5mM Hepes, and 100 U/ml penicillin.
Arginase Activity
Arginase activity was determined by monitoring the formation of [14C]urea from
[guanido-14C]L-arginine. Samples were sonicated in Tris buffer (10mM, ph 7.4) containing
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Triton X-100 (0.4%), leupeptin (10mg/ml) and aprotinin (10mg/ml). Lysates (100μg) were added
to an equal volume of Tris buffer (10mM, pH 7.4) containing MnCl2 (10mM) and arginase was
activated by heating for 10 minutes at 56°C. The arginase reaction was initiated by adding Tris
buffer containing L-arginine (10mM) and [guanido-14C]L-arginine (0.25Ci), and samples were
incubated at 37°C for 30 minutes. Reactions were terminated by adding ice-cold sodium acetate
buffer (250mM, pH 4.5) containing urea (100mM). [14C]Urea was separated from basic amino
acids by Dowex chromatography and [14C]urea formation determined by scintillation counting.
Protein Analysis
VSMCs or blood vessels were lysed in electrophoresis buffer (125mM Tris [pH 6.8],
12.5% glycerol, 2% SDS, and trace bromophenol blue) and proteins separated by SDS-PAGE.
Following transfer to nitrocellulose membranes, blots were blocked with PBS and nonfat milk
(5%) and then incubated with antibodies against arginase I (1:100), cyclin D1 (1:500), cyclin E
(1:500), cyclin A (1:500), p27 (1:300), p21 (1:500), p53 (1:100), or β-actin (1:200). Membranes
were washed in PBS, incubated with horseradish peroxidase-conjugated goat anti-rabbit or rabbit
anti-goat antibodies and developed with commercial chemoluminescence reagents. Protein
expression was quantified by scanning densitometry and normalized with respect to β-actin.
mRNA Analysis
Total RNA (30μg) was loaded on 1.2% agarose gels and fractionated by electrophoresis.
RNA was blot transferred to Gene Plus membranes (Perkin Elmer Life Sciences) and
prehybridized for 4 hours at 68°C in rapid hybridization buffer (Amersham). Membranes were
incubated overnight at 68°C in hybridization buffer containing [32P]DNA probes (1 x 108 cpm)
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for p21 and 18S ribosomal RNA (rRNA). DNA probes were labeled with α-[32P]dCTP using a
random priming kit (Amersham). Following hybridization, membranes were washed, exposed to
X-ray film, and p21 mRNA levels quantified by scanning densitometry and normalized with
respect to 18S rRNA.
Polyamine Production
Polyamine formation was determined by incubating VSMCs with [3H]L-arginine for 24
hours and measuring the intracellular formation of radiolabeled putrescine by thin layer
chromatography.
Cell Cycle Progression
Cell cycle progression and cell viability were assessed by flow activated cell sorting.
VSMCs were serum-starved for 3 days and then treated with serum in the presence or absence of
arginase inhibitors for 24 hours. Cells were then permeabilized, stained with propidium iodide,
and DNA fluorescence measured in a Dickinson FACScan flow cytometer (Franklin Lakes, NJ).
Histograms of DNA content were analyzed using Modfit (Verity Software House, Topsham, ME)
to determine fractions of the population in each phase of the cell cycle.
Statistics
Results are expressed as mean ± SEM. Statistical analyses were performed with the use
of a Student’s two-tailed t-test and an analysis of variance with the Bonferroni post-hoc test
when more than two treatment regimens were compared. P values < 0.05 were considered
statistically significant.