Online Data Supplement
for
Translational Control of JunB, an AP-1 Transcription Factor,
in Activated Human Endothelial Cells
Douglas I Schmid 1,3, Hansjörg Schwertz 1,2,4, Huimiao Jiang 1,3, Robert A Campbell 2,
Andrew S. Weyrich 2,5, Thomas M McIntyre 3, Guy A Zimmerman 3,5 and Larry W
Kraiss 1,3,4
1
3
Division of Vascular Surgery, 2Program in Molecular Medicine,
Eccles Institute of Human Genetics, 4Departments of Surgery & 5Internal Medicine,
University of Utah, Salt Lake City UT
Division of *Vascular Surgery, ‡Department of internal Medicine and the †Program in
Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT, 841125330
DETAILED MATERIALS AND METHODS
Reagents. All culture media and supplements were from Whittaker Bioproducts
(Walkersville, MD) unless otherwise specified. Heat-inactivated fetal bovine serum
(FBS) was supplied by Hyclone (Logan, UT). Rapamycin was obtained from Alexis
(San Diego, CA), tissue culture-grade DMSO was obtained from American Type Culture
Collection (ATCC, Rockville, MD), SB203580 and PD98059 came from Calbiochem (La
Jolla, CA). [-33P]dCTP was obtained from Perkin Elmer (Wellesley, MA), [-32P]dATP
was from ICN (Costa Mesa, CA), and 3H-thymidine from GE Healthcare (Piscataway,
NJ). T4 Polynucleotide Kinase, Murine Malone Leukemia Virus Reverse Transcriptase
(M-MLV RT) and RNasin were obtained from Promega (Madison, WI). Trizol LS, Taq
polymerase, oligo dT, dNTPs, Poly dA, Cot-1 DNA and GeneFilters Named Human
Arrays were obtained from Invitrogen (Carlsbad, CA). Thrombin, cycloheximide and
other chemicals and reagents were purchased from Sigma (St. Louis, MO).
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Polysome profiling. ECs were stimulated with thrombin (2 U/mL) for 2 hours.
Cycloheximide was added to a final concentration of 100 ng/mL for 5 minutes and cells
were then rinsed with cold HBSS containing cycloheximide (100 ng/mL) and scraped
from the dish and pelleted by centrifugation (2000 x g, 5 minutes). The supernatant was
removed and cells were carefully resuspended in 375 L Low Salt Buffer (LSB; 20 mM
Tris, 10 mM NaCl, 3 mM MgCl2, pH 7.4) + RNasin (40 U/mL) + DTT (10 nM) for 3
minutes on ice. 125 L LSB Lysis buffer (LSB containing 200 mM sucrose and 1.2%
Triton-X100) was then added and cells disrupted by pipetting. The mixture was then
transferred to 1.7 mL microfuge tubes and centrifuged (20,000 x g, 1 minute, 4 C) to
remove nuclei and cellular debris. The supernatant was transferred to a new tube
containing 50 L LSB + RNasin (40 U/mL) + DTT (10 nM) and 15 L 5M NaCl. This
mixture was then layered onto prepoured gradients (15-50% sucrose in LSB). Gradients
were centrifuged at 232,000 x g for 90 minutes (4 C) and then separated on a density
gradient flow cell fractionator (Isco Instrumentation) coupled to a spectrophotometer
(254 nM) to obtain ribosomal profiles. Ribosomal fractions corresponding to
monosomes and polysomes were collected into Trizol LS (Invitrogen) and total RNA
isolated according to the directions of the manufacturer.
Translation State Array Analysis (TSAA). Translation state array analysis (TSAA), a
high-throughput method of quantifying the relative distribution of mRNA transcripts
among monosome and polysome subcellular fractions was performed after the method of
Zong et al (Zong et al., 1999) with some modifications (Supplementary Figure 1). To
perform TSAA (Zong et al., 1999) mRNA was isolated from pooled monosome or
polysome fractions obtained as described above from control and thrombin-stimulated
EC yielding four separate samples. Total RNA was reverse transcribed using oligo dT
primers in the presence of [α-33P]dCTP to generate labeled cDNA probes, equal amounts
of which (~10 mcg cDNA/array) were then hybridized to four separate but identical
cDNA arrays (GeneFilters Named Human Array, Invitrogen) and analyzed by
phosphorimaging (Storm Imaging System). After correction for background, array to
array normalization was performed by comparing each element’s intensity to the median
signal intensity for all elements on the array. This normalized intensity was then used to
calculate the translation state (TS) for control and stimulated conditions and then
subsequently the translation index (TLI). Stimulus-dependent redistribution of particular
mRNA species between monosome and polysome fractions out of proportion to any
change in their overall abundance in the cell is taken as evidence of discrete translational
control (Mathews et al., 2000; Zong et al., 1999). This redistribution is reflected by a
derived term, the translation index (TLI), defined as:
 Polysome, stimulated 


Monosome, stimulated 

.
TLI 
 Polysome, control 


 Monosome, control 
The TLI can be calculated for every mRNA species analyzed as long as the relative
quantities in the four ribosomal samples are known. We arbitrarily used TLI thresholds
of ≥ 2.5 as indicating translational upregulation and ≤ 0.4 as indicating translational
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repression in response to a particular stimulus. For a schematic representation of this
process, see Supplementary Figure 1.
RT-PCR. 2 g total RNA was reverse transcribed using MMLV-RT (Promega)
according to the manufacturer’s directions and the cDNA subjected to PCR for -actin
(sense, 5’-TGG AGA AGA GCT ATG AGC TGC CTG-3’; antisense, 5’-GTG CCA
CCA GAC AGC ACT GTG TTG-3’; denaturation step at 94C for 2 min, 25 cycles of
94C for 20 seconds, 60C for 20 seconds, 72C for 45 seconds and a final elongation
step of 72C for 5 minutes) to verify RNA and RT integrity. PCR for JunB then followed
(sense, 5’-CCT GGT GGC CTC TCT CTA CA-3’; antisense, 5'-TGA CCA GAA AAG
TAG CTG CCG CCA CCG-3'; denaturation step at 94C for 2 min, 35 cycles of 94C for
45 seconds, 60C for 45 seconds, 72C for 60 seconds and a final elongation step of 72C
for 5 minutes). Relative differences in mRNA abundance were quantified by the
comparative cycle threshold (2-ΔΔCt ) method (Livak and Schmittgen, 2001).
Nuclear isolation and preparation of nuclear lysates. Experimental media was aspirated
and cells rinsed once in cold PBS + 1 mM PMSF. Fresh PBS + PMSF were then added
and cells scraped and transferred to a centrifuge tube. Cells were pelleted by centrifuging
(14000 rpm, 10 sec, 4 C) and resuspended in 1 mL Lysis Buffer (10 mM HEPES, 10
mM KCl, 1 mM EDTA, 1 mM EGTA, 0.5% Igepal CA-630, 10 g/mL Aprotinin, 100
M Leupeptin, 1 mM PMSF, 1 mM DTT). Cells were incubated on ice for 10 minutes
and then lysed by vortexing (3 minutes) and centrifuged (14000 rpm, 10 sec, 4 C) to
pellet nuclei. The supernatant was removed and nuclear pellet washed twice with 2 mL
Wash Buffer (Lysis Buffer without Igepal CA-630). After the last wash, the supernatant
was removed and 25 L Nuclear Lysis Buffer (10 mM HEPES, 420 mM NaCl, 25%
glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA) used to resuspend pellet. The
pellet was then sonicated for 1 minute, placed on ice for 30 minutes and finally vortexed
for 5 minutes. Nuclear debris was pelleted by centrifugation (14000 rpm, 5 min, 4C)
and supernatant removed to a new tube. Reducing SDS-PAGE buffer was then added to
a final concentration of 0.06 M Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 0.025%
bromphenol blue, and 5% 2-mercaptoethanol. Samples were boiled for 5 minutes,
plunged into an ice bath and centrifuged (14000 rpm, 1 min, 4 C) before loading (~10
µg) onto an acrylamide gel (10% resolving, 5% stacking).
Next-Generation RNA-Sequencing of JunB RNA. RNA was isolated from unstimulated
EC using TRIZOL reagent (Invitrogen). RNA was further cleaned by using a standard
overnight precipitation protocol followed by a rigorous DNase treatment (Turbo DNAfree Kit, Ambion). Post isolation samples were tested on a Agilent Bioanalyzer for
ribosomal RNA integrity indicating good RNA quality. The samples were subsequently
processed for the construction of a Illumina mRNA-seq library and run on an Illumina
Analyzer following the companies protocols.
Western blotting. Extracted proteins (10-20 µg/sample) were separated by SDS-PAGE
and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore,
Bedford, MA) for Western blotting. All steps were performed at room temperature on an
orbital shaker. Primary antibody for JunB (Active Motif) was placed on the membrane
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for 1 hour. After rinsing, the goat anti-rabbit peroxidase-conjugated secondary antibody
(Biosource International) was incubated for 1 hour. Bound antibody complexes were
visualized by enhanced chemiluminescence (ECL, Amersham) and exposure to
radiographic film (Eastman Kodak).
Immunocytochemistry. Stimulated cells were fixed in 2% paraformaldehyde for 20
minutes. All steps were performed at room temperature unless otherwise stated. Cells
were washed with HBSS and permeabilized with 0.5% Triton X-100 for 5 minutes. The
Triton X-100 was removed by rinsing and non-specific binding blocked by incubation
with HBSS containing 10% goat serum (blocking buffer) for 1 hour. Primary antibody
against JunB was then added in blocking buffer overnight at 4 C. Biotin-conjugated
goat-anti-rabbit secondary antibody was then added to the cells for one hour. Slides were
rinsed with HBSS and Alexa 488 streptavidin (in blocking buffer) was added to the cells
for 45 minutes. After additional washing, propidium iodide or TOPRO-3 (Molecular
Probes, Invitrogen) was added to the cells to stain the nuclei.
Cytoplast Preparation. EC were grown on fibronectin coated coverslips until confluent.
2 mL of EC media containing Cytochalasin B (10 µg/mL) was placed into a 30 mL Corex
glass tube. Coverslips were removed from the tissue culture dish and placed, cell side
down, into the medium in the Corex tubes. The cells were placed into an incubator
(37˚C, 5% CO2) for 10 minutes then centrifuged to remove the nuclei (7500 rpm, 60 min,
37˚C, JA 20 rotor). Coverslips were then removed from the Corex tube and placed, cellside up, into a 6 well plate and rinsed 4 times with EC media. Cytoplasts were allowed to
recover for 2 hours in an incubator before use.
SUPPLEMENT REFERENCE
Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402408.
Mathews M, Sonenberg N, Hershey JWB. 2000. Origins and principles of translational
control. In: Sonenberg N, Hershey JWB, Mathews MB, editors. Translational
Control of Gene Expression. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory Press.
Zong Q, Schummer M, Hood L, Morris DR. 1999. Messenger RNA translation state: the
second dimension of high-throughput expression screening. Proc Natl Acad Sci U
S A 96(19):10632-10636.
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Supplement Figure Legends
Supplement Figure 1 – Schematic outline of a typical TSAA experiment.
HUVECs are treated either with vehicle (control) or a specific agonist (experimental) and
lysed. HUVEC lysates are separated into monosome and polysome fractions by sucrose
gradient centrifugation and fractionation. RNA from each of these fractions is isolated,
reverse transcribed and labeled, then used to interrogate cDNA arrays. Signal intensities
are used to calculate a translation state (TS, polysome/monosome signal) for each gene in
each condition. These translation states are then used to calculate a translation index
(TLI). Translational control is implied if the TLI value is greater than 2.5 or less than
0.4.
Supplement Figure 2 – TSAA indicates that a minority of genes in activated EC are
translationally regulated. Confluent human EC were treated with various agonists as
indicated. Polyribosome preparations were then fractionated and pooled into monosome
or polysome samples and analyzed by microarray. TLI values for each array element
were derived by dividing the TS value for activated EC by the TS value for control cells.
In each panel, the TLI histogram is shown above a table of selected gene products whose
TLI suggests translational repression (TLI ≤ 0.4), absence of translational regulation (0.4
< TLI < 2.5) or translational upregulation (TLI ≥ 2.5). . Panel A: histamine (10 mM, 2
hrs). Panel B: insulin (100 µM, 2 hrs). Panel C: LPS (1 μg/ml, 2 hrs). Panel D: TNF-α
(100 u/ml, 2 hrs). Panel E: pooled human serum (20%, 6 hrs).
Supplement Figure 3 – Induction of JunB protein expression is blocked by inhibition of
p38 MAPK in EC activated by thrombin. Human EC were pre-treated with the ERK
pathway inhibitor PD98059 (10 μM), the p38 MAPK inhibitor SB203580 (10 μM), the
mTOR inhibitor rapamycin (10 nM) or the PI3K inhibitor wortmannin (10 nM) for 30
minutes prior to stimulation with thrombin (2 U/ml, 2 hrs). This immunoblot is
representative of three separate experiments.
Supplement Figure 4 – JunB knock-down in EC results in premature senescence. Human
EC were transfected with either scrambled oligo-nucleotide or gene specific oligonucleotide against JunB. Cells were incubated with [3H]-thymidine to evaluate
proliferation. The graph is representative of three independent experiments. Statistically
significant differences (p<0.05) relative to no transfection and scrambled oligo are
indicated by an asterisk.
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