SUPPLEMENTARY METHODS
Quantitative real-time polymerase chain reaction (RT-PCR)
Quantitative PCR was performed under the following thermal conditions: a single
cycle of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s.
The probes for Mrp2, Bsep, AE2 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)
were
as
follows:
Mrp2
5’-CCATTATCCGTGCCTTTGAG-3’
and
5’-ACGACCAAGTTTCCAACCAG-3’
[1];
5’-TTTTCCAGAGGCAGCTATCG-3’
reverse
Bsep
and
5’-ACGACCAAGTTTCCAACCAG-3’
[1];
5’-TAGAGCTAGCGGGTTATGCCTC-3’
forward
and
primer:
forward
reverse
AE2
primer:
primer:
primer:
forward
reverse
primer:
primer:
5’-CGGTGGTATTCAAAGTCTTCC-3’ [2]; GAPDH (as a housekeeping-gene)
forward primer: 5’-ACAGCAACAGGGTGGTGGAC-3’ and reverse primer:
5’-TTTGAGGGTGCAGCGAACTT-3’.
After
normalization
of
the
GAPDH
expression, the expressed mRNA level in each sample was expressed relative to the
control values using the 2-ΔΔCt method [1, 3].
Western blot analysis
Proteins (100 μg protein each) were dissolved in sample buffer and loaded onto a
7.5% sodium dodecyl sulfate polyacrylamide electrophoresis gel with a 4% stacking
gel. The proteins were transferred to polyvinylidene fluoride membranes (Millipore)
after electrophoresis. The membranes were blocked and incubated overnight at 4°C
with the following primary antibodies concentrations: Mrp2 (1:200, Santa Cruz
Biotechnology), Bsep (1:400, Abcam), AE2 (1:200, Santa Cruz Biotechnology) and
Mrp4 (1:200, MyBioSource) β-actin (1:500, Santa Cruz Biotechnology). Signals
were detected using an enhanced chemiluminescent ECL kit (Super Signal, Thermo).
Bands were quantified by densitometry (Image J software). The protein levels were
expressed relative to the control values after normalizing the individual protein bands
to a membrane protein band as an internal reference.
High-performance liquid chromatography (HPLC) analysis of RIF in bile
The bile samples contained 200 μg of ascorbic acid/ml as an antioxidant. The HPLC
system used was an Agilent Model 1200 series (Agilent, USA). Briefly, a Hypersil
ODS2 C18 column (4.6 mm inner diameter × 250 mm, 5 μm, Elite, China) was
maintained at 40.0°C with mobile phase: a mixture of acetonitrile–monosodium
phosphate (0.1 M) (30:70, v/v, PH=5.2) at a flow rate of 1.0 ml/min and with UV
detection at 333 nm. Bile samples were diluted with mobile phase and prepared using
a single-step precipitation process centrifuged at 10,000 g for 15 min; the aliquots
were loaded into the autosampler tray, and in volumes of 20 μl, they were injected
into the chromatograph. The RIF concentration was calculated with a reference for
standard RIF samples (Purity ≥97%, National Institute for the Control of
Pharmaceutical and Biological Products, China).
References
1. Miura T, Kimura N, Yamada T, Shimizu T, Nanashima N, Yamana D, et al.
Sustained repression and translocation of Ntcp and expression of Mrp4 for cholestasis
after rat 90% partial hepatectomy. J Hepatol 2011; 55(2): 407-14.
2. Banales J M, Arenas F, Rodriguez-ortigosa C M, Saez E, Uriarte I, Doctor R B, et
al.
Bicarbonate-rich
choleresis
induced
by
secretin
in
normal
rat
is
taurocholate-dependent and involves AE2 anion exchanger. Hepatology 2006; 43(2):
266-75.
3. Ruiz M L, Villanueva S S, Luquita M G, Ikushiro S, Mottino A D, Catania V A.
Beneficial effect of spironolactone administration on ethynylestradiol-induced
cholestasis in the rat: involvement of up-regulation of multidrug resistance-associated
protein 2. Drug Metab Dispos 2007; 35(11): 2060-66.
SUPPLEMENTARY FIGURES
Supplementary figure 1.
Supplementary figure 1. Procedure for intrabiliary retrograde fluxes experiment.
After intrabiliary retrograde fluxes with DIDS or NPPB, RIF was continuously
infused through the iliac vein. Twenty minutes later, bile was collected during the
15-min experimental period to determine bile flow and biliary compositions.
Supplementary figure 2.
Supplementary figure 2. Effects of RIF on serum levels of ALT and TBA.
Rats were orally administered different doses of RIF (50, 100 and 250 mg/ml) daily
for 1, 3 and 7 days. Data from six rats are shown as the mean ± SD. *P <0.05 vs. the
control value.
Supplementary figure 3.
Supplementary figure 3. Effects of RIF on Mrp4 protein expression in hepatobiliary
transport systems.
Rats were orally administered RIF (100 mg/kg) daily for 1, 3 and 7 days. The
methods used for the preparation of the liver crude plasma membrane fractions and
Western blotting are described in the Materials and methods section. (A) Protein
staining with Coomassie Brilliant Blue. An arrow indicates a protein band used as an
internal reference for protein normalization. Lane Ma, molecular mass marker
proteins. (B) The Western blotting results for Mrp4. Data are representative of 3
independent experiments. Bands were quantified with a scanning densitometer, as
described in the Materials and methods section. The protein levels on days 1, 3 and 7
are expressed relative to the control values. Data from three rats are shown as the
mean ± SD. **P <0.01 vs. the control value.