Download Supplementary Methods

On-line Supplemental Methods:
Western blotting - This procedure was carried out as described in.11 The antibodies used for the
immunoblots were: total-ACC and phosphoS79-ACC from Upstate/Millipore (Billerica, MA), totalAMPK- and phosphoT172-AMPK- from Cell Signaling (Beverly, MA), Bax and Bcl-2 from Santa
Cruz Biotechnology (Santa Cruz, CA). The primary antibodies were all diluted in 3% BSA/TBS-T at a
ratio of 1:1000 and incubated on the membranes at 4°C rocking overnight. A donkey anti-rabbit HRPconjugated secondary antibody from Amersham (Piscataway, NJ) was used at a ratio of 1:10,000 for 1 h
at room temperature. ECL from Pierce (Rockford, IL) was used as the HRP substrate.
Real-time PCR for assessment of mRNA levels - Total RNA from cells grown on a 6 well plates was
extracted with TRIAZOL and 2 g of the total RNA were subjected to the reverse transcriptase reaction
using MMLV with random decamers as the primers, all products were from Invitrogen and used as per
their instructions. The levels of mRNAs and the PCR-product were assessed by real-time PCR using
Cepheid’s Smart Cycler (Hamburg, PA) with melting point analysis. Real Time PCR samples were mixed
with SYBR Premix ExTaq Master Mix (Takara Biotechnology, Inc.; Shiga, Japan) and gene-specific
primers.
The
following
primers
were
used
to
detect
cDNAs:
CyclophilinA;
5'-
GGTGACTTCACACGCCATAA-3' (sense) and 5'-TGTCCACAGTCAGCAATGGT-3' (anti-sense), and
SPTLC1; 5’-AAGGGATTACAGGCATCACG-3’ (sense), 5’-CTCCCAGGACTCCAAATGAA-3’ (antisense). All primers were designed from bovine sequences with the use of the Primer3 on-line tool
(http://frodo.wi.mit.edu/primer3/) and purchased from Operon (Huntsville, AL). The relative Ct
method was used to calculate fold change with CyclophilinA used as the normalizing gene.
NF-B Reporter Gene Assay - Transactivation of NF-B was assessed by dual infection with a NF-Bfirefly-luciferase expressing adenovirus and a constitutive CMV-renilla-luciferase expressing adenovirus
at titers of roughly 100-200 x 106 PFU/ml. Both were made in-house from plasmid sequences from the
Mercury Profiling System (Clontech/Takara). Forty-eight hours post infection the PCs were treated as
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indicated in the experiment. After treatment, the cells were placed on ice, washed 2 times with cold PBS
then lysed with 1X passive lysis buffer (Promega, Madison, WI). The two luciferase signals were
assessed from the lysates with a dual luciferase assay kit (Promega) and a BioOrbit 1251 Luminometer
(LKB Wallac; Turku, Finland). All data are representative experiments of at least three independent
repetitions and are shown as mean ± standard error.
Oxidative stress measurements:
Dichlorofluorescein (DCF) Fluorescence - 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein
diacetate, acetyl ester (CM-H2DCFDA; Molecular Probes) is a cell permeable nonfluorescent compound
that is trapped in cells when cleaved by intracellular esterases and is then converted into the fluorescent
product 2’,7’-dichlorofluorescein (DCF) in the presence of H2O2 and other ROS molecules. Thus, it
allows for the measurement of oxidative stress within cells. This procedure was carried out as described
in.11
8-Isoprostane Measurement - The isoprostanes, products of lipid peroxidation, were measured by ELISA
according to the manufacturer’s instructions (StressGen; Ann Arbor, MI). Essentially, PCs were grown in
6-well plates, 1 mL of media per well, and treated as described in the specific experiment. After the
experiment, the media were collected in Eppendorf tubes and frozen down in liquid N2. With this kit, 50100 l of undiluted media was sufficient to measure the isoprostanes. The data were typically normalized
to total protein content per well.
Electron-Spin Resonance Spectroscopy (ESRS) - ESRS is a method for measuring oxidative stress based
on the properties of a particular compound which acts as an electron spin-trap. Essentially, the “spin-trap”
is a chemical that under baseline conditions does not have any paramagnetic properties, but when in the
presence of ROS (e.g. superoxide) an adduct forms between the spin-trap and the ROS which gives the
spin-trap a paramagnetic property that is measurable by nuclear magnetic resonance (NMR).
The oxidase activity of NAD(P)H oxidase was measured by this technique as follows: PC grown in
100mm dishes were treated as indicated for a given experiment. After treatment, the cells were placed on
ice and washed three times with cold PBS. Two ml of HES buffer (10mM hepes, 1mM EDTA, and
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250mM sucrose, final pH 7.4) was given to each plate, the cells were scraped, collected, sonicated (30%
duty cycle, 10 strokes), and then centrifuged at 100,000 x g in a Sorvall Ultracentrifuge OTD75B
(DuPont, Newtown, CT) for 1 h to separate the membrane fraction, containing the membrane-bound
“active” NAD(P)H oxidase, from the cytosolic fraction. To the membrane fraction (pellet) 650 l of HES
buffer were added and the mixture dounce homogenized. Five l of the membrane mixture were taken for
protein determination by BCA assay. The rest of the membrane mixture was divided into 2 – 300 l
aliquots and transferred into two separate Eppendorf tubes. To initiate ROS generation by NAD(P)H
oxidase both of the paired samples were incubated with 50mM DMPO (5,5-dimethyl-1-pyrroline Noxide, Sigma), as the electron spin-trap, 100M diethyldithiocarbamate, 300M NAD(P)H (as the
substrate for NAD(P)H oxidase) in PBS. One of the paired samples was given 10M of the NAD(P)H
oxidase specific inhibitor diphenyleneiodonium chloride (DPI) to determine if the ROS activity was
indeed from NAD(P)H oxidase. The reactions were then incubated for 1hr at 37°C to allow enough time
for ROS-DMPO (paramagnetic) adduct formation. ESR spectra were recorded using a Bruker EMX
spectrometer at 9.78 GHz, 20mW with a modulation amplitude of 1.0 G for 10 min.
Total ceramide and diacylglycerol (DAG) measurement - Total DAG and ceramide were quantified from
the total lipids by using sn-1,2-diacyglycerol kinase (Calbiochem) in the presence of
Elmer; Boston, MA) to convert ceramide to ceramide-1-32P -phosphate and DAG to
32
P-ATP (Perkin-
32
P-phosphatidic
acid.48 DAG and C16-ceramide standards (Avanti) were also included at concentrations of 10 g each for
the calculation of specific activity. After completion of the reaction, the lipids were chloroform extracted
and chromatographed on silica gel 60 thin layer chromatography (TLC) plates (Whatman, Clifton, NJ)
with a solvent mixture composed of cholorform-pyrimidine-formic acid (65:35:7.5;v/v/v) followed by
chloroform-methanol-acetic acid (65:15:5;v/v/v). The TLC plates were then subjected to autoradiography
and the radiographic bands corresponding to ceramide-1-phosphate were excised, transferred to
scintillation vials with 5 ml Ecoscint (National Diagnostics, Atlanta, GA), and quantified by scintillation
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counting on a Rack-Beta Liquid Scintillation Counter (LKB Wallac; Turku, Finland). Densitometric
analysis of the autoradiogram was sufficient for the quantitation of DAG.
Measurement of 3H-serine incorporation into sphingolipids - Approximately ~85% confluent cells grown
in 6-well Primaria dishes were incubated in palmitate or BSA containing medium supplemented with 3Hserine (Perkin-Elmer) (0.4 Ci/ml), for 24 h. Lipids were extracted using the Bligh-Dyer method. TLC
separation of lipids was performed using a modified method based on van Echten-Deckert.49 Briefly,
dried lipids were re-suspended in chloroform:methanol (1:1;v/v) containing 10 l each of sphingolipid
standards ceramide, dihydroceramide, sphingosine, sphinganine, and phophatidylserine (all from Avanti
Polar Lipids; Alabaster, AL) at a concentration of 1 g/ml. Lipids were spotted onto the origin of a silica
gel-60 TLC plate. A solvent system of chloroform:methanol:3.5 N ammonium hydroxide (85:15:1;v/v/v)
was added to a TLC chamber and allowed to saturate with vapors using a filter paper wick. Sphingolipid
bands were visualized with iodine vapors, excised and quantified by liquid scintillation counting.
Serine palmitoyltransferase activity assay - Cells were grown to ~80 - 85% confluence and treated as
indicated for 24 h. They were washed once with ice-cold PBS, and then harvested in a buffer containing
20mM Tris-HCl (pH 7.4), 50mM NaCl, 30mM NaPPi, 1mM EDTA, 0.25mM sucrose, 30mM NaF, 1mM
Na3VO4, 1g/ml leupeptin, 1g/ml pepstatin, 1mM PMSF, 2mM dithiothreitol (DTT), 0.4mg/ml
digitonin, and 5mM EGTA. The mixture was homogenized on ice with a dounce homogenizer and
centrifuged in a Sorvall Ultracentrifuge OTD75B (DuPont) at 250,000 xg for 60min at 4°C. The
supernatant was removed, and the membrane pellet re-suspended in 300 l of a buffer containing 10mM
Tris-HCl (pH 7.4), 1mM EDTA (pH 7.4), and 1mM DTT. SPT activity was measured in isolated
microsome fractions as described by Williams et al.
50
and Dickson et al.,51 with minor modifications. In
brief, activity was assayed in a total volume of 0.1 ml of a 100mM hepes/KOH (pH 8.3) buffer containing
5mM DTT, 2.5mM EDTA with 50l of the microsomal sample. The reaction was initiated by the addition
of 200M palmitoyl-CoA and 3H-serine, and was carried out at 37 °C for 10 min under light-protected
conditions. It was terminated by the addition of 0.2 ml of 0.5 N NH4OH and 1.5 ml CHCl3:methanol (1:2;
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v/v). After this, sphinganine (25 l of 1mg/ml in ethanol) was added as a carrier, followed by the addition
of 1 ml chloroform and 2 ml 0.5N NH4OH. Samples were vortexed vigorously and centrifuged to separate
the layers. The aqueous upper phase was washed twice with 2 ml 200mM NaCl to remove any
unincorporated radiolabeled serine. From the chloroform layer 0.5 ml was transferred into a scintillation
vial, air-dried, resuspended in 5 ml scintillation fluid, and quantified by liquid scintillation counting.
Enzyme activity was expressed as pmol 3-ketosphinganine/mg protein/min.
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