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Supplementary Information
Methods
Cell culture
All cells were cultured in RPMI 1640 (12-702F; Lonza, Basel, Switzerland) containing 10%
fetal bovine serum (FBS) with penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) in 5%
CO2 air humidified at 37°C.
Western Blot
Whole cell lysates were prepared by adding 1.5 mL cell lysis buffer (10 nM Tris-HCl, pH
8.0; 150 mM NaCl; 1 mM phenylmethylsulfonyl fluoride, and 1% Triton X-100) to cells
grown to 80% confluence in T175 tissue culture flasks. Sixty micrograms of protein were
loaded onto 4–20% Novex Tris glycine gradient denaturing polyacrylamide gels (Invitrogen)
in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) running buffer,
containing 1 g/L SDS, 3 g/L Tris-base, and 14.4 g/L glycine. The proteins were transferred
onto polyvinylpyrolidine difluoride membranes by electrophoresis. Once transferred, the
membranes were blocked in Blotto [5% dry milk resuspended in Tris-buffered saline (0.9%
NaCl, 10 mmol/L Tris pH 7.4, and 0.5% MgCl)] at room temperature for 1 hour. Primary
antibodies and dilutions used were anti-SMO (sc-166685; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) 1:250, anti-SHH (#2287, Cell Signaling Technology, Danvers, MA, USA)
1:1000, anti-PTCH (sc-6149; Santa Cruz Biotechnology) 1:1000, anti-GLI-1 (sc-271075;
Santa Cruz Biotechnology) 1:100, anti-GLI-2 (sc-28674; Santa Cruz Biotechnology) 1:100,
anti-Caspase8 (sc-81662; Santa Cruz Biotechnology) 1:200, anti-Caspase9 (sc-56073; Santa
Cruz Biotechnology) 1:200, anti-Caspase3 (#9662S, Cell Signaling Technology) 1:200, antiBax (ab32503; Abcam, Cambridge, MA, USA) 1:100, anti-Bcl2 (sc-130308; Santa Cruz
Biotechnology) 1:100, and anti-Bak (ab1080; Abcam) 1:100. All were incubated with the
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membrane at 4°C overnight and the membrane was washed three times, 10 min each time,
before the addition of goat anti-rabbit secondary antibody conjugated with horseradish
peroxidase at 1:1,000 dilution for 1 hour. Immune complexes were identified with enhanced
chemiluminescence western blotting detection reagents (PierceTM ECL Western Blotting
Substrate; Thermo Scientific, IL, USA). Membranes were stripped and subsequently blotted
with anti-β-actin antibody as a loading control.
Transfection of RNA oligonucleotides
Cells were transfected with siRNAs using the Lipofectamine 2,000 reagent (Invitrogen)
according to the manual’s instructions after cells were grown to 80% confluence.
Transfection was repeated three times for 3 days. After treatment, cells were stimulated for
western blot screening.
Human sample and immunohistochemical staining
Three micrometer thick sections were cut from formalin-fixed and paraffin-embedded tissue
and mounted on glass slides coated with silane (Matsunami, Tokyo, Japan), deparaffinized in
xylene, dehydrated in ethanol, and dried in a microwave oven with 10 mM sodium-citrate
buffer (pH 6.0) for antigen retrieval. The primary antibodies used were a rabbit polyclonal
anti-Gli2 antibody (ab26056; Abcam) at a dilution of 1:200, a rabbit polyclonal anti-Ihh
antibody (Santa Cruz Biotechnology) at a dilution of 1:25, and a goat polyclonal anti-Patched
antibody (sc-6149; Santa Cruz Biotechnology) at a dilution of 1:50. Slides were incubated for
1 hour and were developed using an Enhanced Dab detection kit (LP kit; Thermo Scientific,
Fremont, CA, USA) or goat ABC kit (Santa Cruz Biotechnology). Endogeneous peroxidase
activity was blocked with 3% hydrogen peroxide in methanol for 20 minutes. After washing
with phosphate-buffered saline (PBS) three times, the sections were incubated with the
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enzyme-conjugated secondary antibody. Hematoxylin was used for counterstaining. The
proportion of expression in the tissue was scored as 0 (0–5%), 1 (6–33%), or 2 (34–100%) by
the same experienced pathologist (SH Park). The median cut-off value of the summed scores
of intensity and proportion was 1.
Proliferation Assay (MTT assay)
3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) assays
were
performed as follows. In brief, 1  104 cells per well were incubated in 100 μL of medium
containing 0.5% FBS with 6 μM cyclopamine, solvent, or medium only for 72 hours. Finally,
20 μL/well of CellTiter96 solution (Promega, Madison, WI, USA) were added for 1 hour and
plates were read on a Wallac-1420 plate reader at an absorbance of 490 nm (Perkin-Elmer,
Boston, MA, USA). All experiments were set up in triplicate to determine means and SDs.
Fluorescence-Activated Cell Sorting Analysis
HuCCT-1 cells were cultured and treated with the HH signal inhibitors at 10 μM for 48
hours. The extent of apoptosis was measured by fluorescence-activated cell sorting (FACS)
after fluorescein isothiocyanate (FITC) annexin-V staining using a FITC Annexin V
Apoptosis Detection Kit II (BD Biosciences, Chicago, IL, USA).
Coculture Proliferation Assay
The proliferation of cultured CC cells (HuCCT-1, SNU-308, SNU-245, and SNU-1196) was
assayed using a co-culture system consisting of a six-well plate and cell culture insert
chambers (BD Biosciences). Lx-2 cells (5  104) were seeded in the upper chamber and CC
cells (2  105) were seeded in the lower chamber. To document proliferation efficiency, the
chambers were incubated in a 5% CO2 air-humidified atmosphere at 37°C. Cell growth was
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analyzed after 24 hours by adding 100 μL/mL of WST-1 solution (Takara Bio Inc, Shiga,
Japan) for 4 hours and measuring the absorbance at 450 nM using an ELISA reader (Bio-Tek,
Winooski, VT, USA). The proliferation of LX-2 was assayed using a co-culture system
consisting of a six-well plate and cell culture insert chambers (BD Biosciences). HuCCT-1
cells (1  104) were seeded in the upper chamber and LX-2 cells (2 x 105) were seeded in the
lower chamber. The medium in each well was changed daily. Cell growth was analyzed after
24 hours by adding 100 μL/mL of WST-1 solution (Takara Bio Inc) for 4 hours and
measuring the absorbance at 450 nM using an ELISA reader (Bio-Tek).
Wound Healing Assay
For the measurement of cell migration, wound healing assays were performed with HuCCT1/SNU-308 and Lx-2 cells. Cells (1  104) were seeded in 6-well plates and grown into a
monolayer for 24 hours. A 200-ml pipette was used to make a scratch through the monolayer
and the medium was replaced with RPMI 1640. RPMI containing 7% FBS was used as a
control. After adding RPMI containing 7% FBS and 10 μM of each agent, cells were grown
for 24 hours. Cells were washed with PBS twice and fixed in methanol for 3 minutes. Wound
widths were measured using IMT iSolution Lite ver. 9.1 (IMT i-Solution,Vancouver, BC,
Canada).
Coculture invasion assay
Biocoat Matrigel-coated invasion chambers (BD Biosciences) were used to examine cell
invasiveness. In brief, 1  104 HuCCT-1 cells in 500 μL serum-free medium were added to
the upper chamber. Medium containing 10% FBS or concentrated Lx-2 cultured medium was
added to the lower chamber. Serum-free medium was added to the lower chamber of control
wells. The cells were allowed to invade the Matrigel for 24 hours at 37°C in a 5% CO 2
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atmosphere. The non-invading cells on the upper surface of the membrane were removed
with a cotton swab. Cells were fixed in methanol, stained with 1% toluidine blue, and airdried. Invading cells in each membrane were counted 10 times and averaged at 40
magnification.
Cytokine array
HuCCT-1 and LX-2 cells (4  105) were cultured using RPMI 1640 with 10% FBS at 37°C
with 5% CO2 for 3 days. After 24 hours, cells were incubated in RPMI 1640 with 1% FBS for
24 hours, treated with cell lysis buffer, and centrifuged at 13,000 rpm for 1 minute to produce
the supernatant. Aliquots containing 50 µg of protein were examined using a RayBio®
Human cytokine antibody array chip (Raybiotechm, Norcross, GA, USA). The chip
antibodies were basic fibroblast growth factor (bFGF), epidermal growth factor (EGF),
fibrobast growth factor-7 (FGF-7), hepatocyte growth factor (HGF), insulin-like growth
factor (IGF-1), interleukin-6 (IL-6), platelet-derived growth factor-BB (PDGF-BB),
transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and
human sonic hedgehog (SHH-h). The relative expression levels of these cytokines were
calculated by densitometry. The positive control was used to normalize the results from
different membranes being compared. Each test was repeated four times.
Size measurement of xenograft
Tumor growth was monitored biweekly by measuring tumor diameters with a caliper. Tumor
volume was calculated using the formula (a  b  c) ÷ 2, where a and b are the short and long
diameter of the tumors, respectively, and c is the thickness.
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RNA quantification of xenograft
Total RNAs were extracted from fresh-frozen tumor tissue using the easy-BLUETM Total
RNA Extraction kit (Intron Biotechnology, Sungnam, Korea) according to the manufacturer's
protocol. Reverse transcription polymerase chain reaction (RT-PCR) was performed using an
RNA PCR kit (AMV) ver. 2.0 (Takara, Shiga, Japan) using 1 μg of total RNA. Primer
sequences were as follows: Gil1 transcript, 5-CTCCCGAAGGACAGGTATGTAAC-3 and
5-CCCTACTCTTTAGGCACTAGAGTTG-3;
Gil2
TGGCCGCTTCAGATGACAGATGTTG-3
CGRRAGCCGAATGTCAGCCGRGAAG-3';
transcript,
and
SMO
55-
transcript,
5-
GGAGAGGAGCCATACTGCCC-3 and 5-TCAACCAGCCACAGGTTGG-3; and GAPDH
(control),
5-ATCTTCCAGGAGCGAGARCCC-3
and
5-
CGTTCGGCTCAGGGATGACCT-3. The PCR reaction mixture was resolved by 1.2%
agarose gel electrophoresis and stained with ethidium bromide. Digital images were saved in
a computer in Joint Photographic Experts Group (JPEG) format. ImageJ 1.47 (downloaded
from http://rsbweb.nih.gov/ij/download.html) was used to analyze each band according to the
manual.
Xenograft proliferation
Cell proliferation in tumor tissue was measured by the Brd-U cell proliferation assay. Three
percent hydrogen peroxide (H2O2) solution was used to suppress endogenous peroxidase
activity. The tissue was incubated with mouse anti-Brd-U (#6326; Abcam) at 1:50 dilution for
12 hours at 4°C. After removing antibodies, tissues were incubated with the DAKO REALTM
EnVisionTM Detection system (Millipore, Billerica, MA, USA). The level of cell proliferation
within cancer tissue was quantified by measuring Brd-U-incorporated cells in 10 randomly
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selected microscopic fields at 400 magnification.
Apoptosis of xenograft
Apoptosis in tumor tissue was investigated by TUNEL staining using ApopTag® Peroxidase
In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA) after fixing fresh tissue in
10% formalin for 2 days. After removing the antibody, tissues were incubated with the
aforementioned DAKO REALTM EnVisionTM Detection system. Terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL)-positive cells were counted in 10 different
high-power fields at 400 magnification. The apoptotic index was calculated and represented
as a percentage of TUNEL-positive cells.
Quantitative evaluation of the necrotic area of xenograft
H&E staining was performed to assess the morphological features of necrotic tumor regions.
A BX 51 microscope with an attached DP 70 camera (Olympus, Tokyo, Japan) was used to
scan whole tissue sections. Digital images were saved in a computer in JPEG format for the
measurement of percentage necrosis. Aperio ImageScope V11.0.2.725 (Aperio Technologies,
Vista, CA, USA) was used to draw boundaries around the entire tumor region and analyze the
viable area, and to measure variables including the number of positive and negative cells.
Necrotic area/total area % was calculated by [(total number – number of negative) ÷ total
number]  100.
Microvessel density (MVD) of xenograft
Immunohistochemical staining for anti-CD 31 (#28364, 1:200 dilution; Abcam) on paraffinembedded sections was performed using the aforementioned REALTM EnVisionTM Detection
system. CD 31-positive microvessels in the most intense vascular area of tumor tissues were
counted in 10 different high-power fields at 400 magnification. Intratumoral mean MVD
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was expressed as number of microvessels/mm2.
Supplementary Table
Half-maximal inhibitory concentration (IC50, μM) values for HH inhibitors in cell lines.
SNU 245
SNU 308
SNU 1079
SNU 1196
HuCCT-1
Lx-2
Cyclopamine
Tomatidine
50.76
11.40
56.26
80.82
12.56
4.87
77.40
60.00
37.78
151.89
61.44
71.70
GANT-58
GANT-61
GDC-0449
86.51
72.35
78.13
102.45
103.88
8.33
159.34
95.56
118.31
474.57
65.23
64.31
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Supplementary Figure Legends
Supplementary Figure 1: Example of immunohistochemical staining of a human intrahepatic
cholangiocarcinoma specimen. (A) Indian Hedgehog; (B) Patched. 400 magnification.
Supplementary Figure 2: MTT assay using specific Hedgehog inhibitors (GDC-0449, GANT
58, GANT 61) in cholangiocarcinoma cells (HuCCT-1, SNU-308, SNU-1196) and hepatic
stellate cells (Lx-2). (A) Proliferation of HuCCT-1 cells was not suppressed by specific
Hedgehog inhibitors. (B) Proliferation of SNU-308 cells was not suppressed by specific
Hedgehog inhibitors. (C) Proliferation of SNU-1196 cells was not suppressed by specific
Hedgehog inhibitors. (D) Proliferation of Lx-2 cells was suppressed by GANT 58; the IC50
was 8.33 μM.