Supplementary Material and Methods (for online publication only)
Morphological Studies—For light microscopy, pieces of liver were fixed in 4%
paraformaldehyde, embedded in paraffin, and 3-m-thick sections stained with hematoxylin
and eosin. Frozen sections of formalin-fixed liver (5 m) were stained with Oil Red O and
counterstained with Giemsa. For cell proliferation analysis, livers were processed for
immunohistochemical localization as described previously,[23] using antibodies raised
against BrdUrd (Becton Dickinson, San Jose, CA). Histological analysis and image
processing were carried out using Leica DMRE microscope equipped with a Spot digital
camera. Images were taken at x20 and x40 magnification and captured at 1315 x 1033 pixels.
Montages of images were prepared with the use of Photoshop 5.0 (Adobe, Mountain View,
CA).
Immunostaining—Immunohistochemical staining was performed by an avidin-biotinylated
peroxidase complex (ABC kit, Vector Laboratories, Burlingame, CA) or by the peroxidaseanti-peroxidase method as described.[1, 2] Negative controls consisted of staining with
normal rabbit serum instead of antibodies or by omitting the primary antibodies. Slides were
counterstained with hematoxylin. Immunohistochemical localization of ACOX1 proteins in
tissues fixed in 10% formalin or 4% paraformaldehyde was accomplished by using
monospecific polyclonal antibodies.[1, 2]
Catalase Histochemistry—For cytochemical localization of catalase, tissues were fixed in
1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 4 h at 4 °C, washed
overnight with 0.1 M cacodylate buffer (pH 7.4), and cut into 60-mm sections with a
vibratome. These sections were stained for catalase with alkaline 3,3-diaminobenzidine
substrate, postfixed with 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4) as
described.[1, 2] Semithin sections, with or without toluidine blue counter stain, were
examined by light microscopy.
Hepatocyte Isolation and Culture—Hepatocytes were isolated from wild-type and ACOX-/mice livers using the collagenase perfusion method.9 Hepatocytes were kept in Standard
William’s Media E modified (Gibco, Carlsbad, CA) at 37°C in a humidified atmosphere
containing 95% air and 5% carbon dioxide. They were plated (4x106) and infected with
Ad/hACOX1a, Ad/hACOX1b or Ad/LacZ. Cells were harvested for RNA and proteins
extraction 48 hr after infection.
RNA Preparation, Northern Blotting, and Real-time-PCR — Total RNA was prepared from
mouse tissues using TRIzol reagent (Invitrogen, Carlsbad, CA or Cergy Pontoise, France).
Human total RNA survey panel was from Ambion (Huntingdon, United Kingdom). RT-PCR
was performed with 2 µg of total RNA with SuperScriptTM III One-Step RT-PCR System
using Platinum Taq DNA polymerase (Invitrogen). Total RNA (20 µg) extracted from liver
was glyoxylated, separated on 0.8% agarose gel, and transferred to nylon membranes. cDNA
probes used for Northern blotting included ACOX1, L-PBE, thiolase, CYP4A10, CYP4A14,
PPAR, all derived from rat.7 GAPDH was used as a probe to evaluate the amount of total
RNA loaded. Quantitative PCR procedure was used for the ACOX, L-PBE, thiolase,
CYP4A1, CYP4A3, PPAR, and GAPDH using the primer sequences in supplementary
Table S1.
Western Blot Analysis—For Western blot analysis, 50 µg of protein from liver extracts were
separated by SDS-PAGE, blotted onto nitrocellulose membrane. Immunoblotting was
performed by employing rabbit anti-human ACOX1, anti-rat L-PBE, anti-rat catalase, or antirat PPAR antibodies as the primary antibodies1, 2 and alkaline phosphatase-coupled goat
anti-rabbit IgG (Bio-Rad, San Diego, CA) as the secondary antibodies.
Chromatin Immunoprecipitation (ChIP) Assays—Liver chromatin immunoprecipitation
assays were performed as described.[9] Liver nuclei were purified and fixed for 30 min in 1%
formaldehyde to cross-link the DNA-binding proteins to cognate cis-acting elements. Nuclear
homogenates were sonicated to shear the chromosomal DNA to an average length of 1000
bp.[9] The primers used for amplification of the PPAR response unit of the mouse L-PBE
promoter region were 5’–AAATTTACCACTGTGACCACTAGATGTC-3’ and 5’AGAGAAAAGCCTAGACCTATGCCTG-3’(AC130214).
Enzymatic assay. the ACOX1 activity was assayed by the fluorimetric based assay (29) in
liver homogenate. The reaction mixture (200 µL) contained 50 mM Tris buffer at pH 8.3, 0.75
mM homovanillic acid, 20 lg ml-1 horseradish peroxidase and 50 µM ofpalmitoyl-CoA as
substrate. All reactions were started by the addition of 20 µM of a 0.625 µM enzyme solution.
Biochemical assays were performed according previously described methods: Very-longchain fatty acids, pristanic acid and phytanic acid (1) and plasmalogens and polyunsaturated
fatty acids (PUFA) (2)
1. Vreken, P., van Lint, A.E., Bootsma, A.H., Overmars, H., Wanders, R.J.
and van Gennip, A.H. (1998) Rapid stable isotope dilution analysis of very-long-chain fatty
acids, pristanic acid and phytanic acid using gas chromatography-electron impact mass
spectrometry. J. Chromatogr. B Biomed. Sci. Applic., 713, 281-287.
2. Dacremont, G. and Vincent, G. (1995) Assay of plasmalogens and polyunsaturated fatty
acids (PUFA) in erythrocytes and fibroblasts.
J. Inherit. Metab. Dis., 18, 84-89.