Supplemental Materials and Methods
Immunohistochemistry. To quantify fibrosis, 5 m sections were stained with
picrosirius red (Sigma) and counterstained with fast green (Sigma). The proportion of
tissue stained with picrosirius red content was assessed by morphometric analysis with
Metaview software (Universal Imaging Corp, Downington, PA) as described(1). For
analysis, 15 randomly chosen, 20 fields were used.
Primary antibodies used were as follows: -SMA (1:500; Abcam 5694), Gli2
(1:2000, Genway Biotech 18-732) and Ki67 (1:750, Novocastra NCLKi67p). Antigens
were demonstrated by diaminobenzidine (DAB) (Dako). Omitting primary antibodies
eliminated staining, confirming specificity. Hepatocytes and bile ductular cells staining
positive for Ki67 and Gli2 were counted. For each slide, 15 randomly chosen, 40x fields
were evaluated. The average number of Gli2 positive bile duct cells was obtained by
dividing the total number of positive cells by the total number of portal tracts. Gli2(+)
hepatocytes was expressed as a percentage of the total number of hepatocytes per
high-powered field. For SMA quantification, 15 randomly selected, 40 fields
(excluding the major bile duct in each portal tract from consideration) were analyzed
with the Metaview software.
DNA replication index was assessed by in vivo nuclear incorporation of 5-bromo2’-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO). BrdU (50 g/g of body weight)
was injected intra-peritoneally 2h before sacrifice. Sections were processed using
mouse anti-BrdU (1:100 dilution; Dako Cytomation M0744). BrdU(+) nuclei in 15
randomly selected, 20 fields were evaluated for the BrdU labeling index. Hepatocyte
BrdU(+) staining was expressed as a percentage of total hepatocyte counted, and
ductular BrdU(+) staining was expressed as the average number of positive cells per
portal tract. For each time point, sections from 4 animals were analyzed.
mRNA Quantification by Real Time RT-PCR. Total RNA was isolated from
tissues using Trizol (Invitrogen, Carlsbad, CA), purified with RNeasy Mini kit (Qiagen,
Chatsworth, CA); genomic DNA contamination was eliminated with DNAse I
(Invitrogen). The purity and concentration of RNA was determined with NanoDrop ND1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Palo Alto, CA). A 260/280
nm absorbance ratio of 1.8-2.1 was always recorded indicating pure RNA samples.
cDNA was synthesized from 2-3 g of RNA using the SuperScript III First-Strand
Synthesis System (Invitrogen), according to the manufacturer’s recommendations for
random hexamers primed cDNA synthesis. Gene transcripts were quantified by realtime RT-PCR (Eppendorf, Mastercycler Real Time PCR). Amplification reactions were
carried out using 20 L of SYBR Green PCR Master Mix (Applied Biosystems) and 5 L
of template cDNA. The Sequence Detection System was programmed as follows: a 10
min denaturation at 95ºC, followed by 40 cycles of 15 sec at 95ºC and 1 min at 60ºC,
extension of 72ºC for 10 min, and a melting curve program (60-95ºC, heating rate of
0.2ºC/sec). All samples were analyzed in triplicate. Gene expression levels were
normalized to the reference gene S9, according to the Ct method(2). Sequences of
primers are listed in the Supporting documents (Supplementary Table 1 online).
References
1.
Yata Y, Gotwals P, Koteliansky V, Rockey DC. Dose-dependent inhibition of
hepatic fibrosis in mice by a TGF-beta soluble receptor: implications for antifibrotic
therapy. Hepatology. 2002;35(5):1022-30.
2.
Janovick-Guretzky NA, Dann HM, Carlson DB, Murphy MR, Loor JJ, Drackley
JK. Housekeeping gene expression in bovine liver is affected by physiological state,
feed intake, and dietary treatment. J Dairy Sci. 2007;90(5):2246-52.