Supporting Information
Supporting Figure 1. A) Specificity of siRNAs targeting HER2. Two clones of siRNAs targeting
HER2 (siHER2 #1 & siHER2 #2, respectively) alone with siRNA with scrambled sequences (siNS)
were used to exclude the off-target effect of RNA interference suppression of phosphorylation of
both ERBB3 and the downstream Akt. Forty-eight hours after transfection, HCC cells were
harvested and subjected to immunoblotty assays for the expression of phosphorylated ERBB3 and
phosphorylated Akt, respectively. B) Specificity of siRNAs targeting ERBB3. Two clones of
siRNAs targeting ERBB3 (si ERBB3 #1 & si ERBB3 #2, respectively) alone with siRNA with
scrambled sequences (siNS) were used to exclude the off-target effect of RNA interference
suppression of phosphorylation of ERBB3 and the downstream Akt. Forty-eight hours after
transfection, HCC cells were harvested and subjected to immunoblotty assays for the expression of
phosphorylated ERBB3 and phosphorylated Akt, respectively. C) The effects of silencing HER2 or
ERBB3 on the proliferation of HCC cells. Two clones of siRNAs targeting HER2 (siB2, #1 and #2))
and two clones targeting ERBB3 (siB3, #1 and #2) were used to silencing HER2 and ERBB3
expression, respectively. The representative results show that are derived from HepG2 cells. Though
silencing HER2 or ERBB3 expression suppressed the phosphorylation of ERBB3 (A) and
downstream activation of Akt pathways (B), there was no obvious effect on cell proliferation (C).
Supporting Figure 2. Effects of gefitinib and lapatinib on the proliferation and viability of HCC
cells. A total of 1500 cells were seeded onto each well in 96-well plates 24 hours before treatment
with gefitinib or lapatinib. Serial concentrations of gefitinib and lapatinib were administered to
HCC cells and then the cell viability was analyzed with XTT assays 48 hours after treatment. The
experiments were conducted twice in triplicate. The IC50 values of gefitinib were ranged from 29
M (Huh7 cells) to >150 M (Tong cells), while the IC50 values of lapatinib were ranged from 17
nM (Huh7 cells) to 53 nM (Hep3B cells). Lapatinib apparently has more potent suppressive effects
on cell proliferation and viability of HCC cells than gefitinib.
Supporting Figure 3. Enhancement of cell motility by activation of NRG1/ERBB3 signaling.
Huh7, SK-Hep1, and HepG2 cells (A, B and C, respectively) were starved and wound was created
with a p-200 pipette tip 16 hours after seeding followed by maintained in the medium supplemented
with 0, 1, 10, and 50 ng.ml of NRG1. A dose dependent increase of cells migrating into the wound
domains was noted. Images were attained 0h, 24h and 48h after wound creation and only those
taken 0h and 48h are shown. Magnification: 40× for the left and middle panels; 100×, the right
panel. D) Numbers of cells migrated into the wound domains per 100× magnified field. The
experiments were conducted twice in duplicate.
Supporting Figure 4. Immunoblotting of ERBB3 of xenografts derived from Huh7 cells
transduced with shRNA targeting luciferase gene (C) or shRNA targeting ERBB3 (B3). Two
xenograft tumors of each group were examined.
Materials and Methods
Quantitative real-time polymerase chain reaction
Total RNA was extracted from tissues and HCC cells using Trizol total RNA isolation reagent
(Invitrogen), according to the manufacturer’s instruction and qRT-PCR was conducted using the
ABI PRISM 7000 sequence detection system (Applied Biosystems) as previously described.22.
Equal amounts of RNA were used for all qRT-PCR reactions, which were performed in triplicate,
and 18S ribosomal RNAs and the -actin mRNA were used as internal controls.
Immunoblotting analysis and Immunohistochemical staining
Cells were homogenized and lysed in a buffer containing 50 mM Tris-HCl pH 8.0, 120 mM NaCl,
0.5% NP-40, 0.25% Na deoxycholate, 1 mM DTT, 2 mg/ml aprotinin, and 2 mg/ml leupeptin.
Thirty micrograms of the lysate proteins were subjected to electrophoresis followed by
immunoblotting analysis using antibodies against the target proteins. A loading control of detection
of -actin was included for all the immunoblots. The protein lysate was resolved in 8% SDS
polyacrylamide gels, transferred to a nylon membrane (PVDF, Schleiche & Schuell, Einbeck,
Germany), and then incubated with primary antibodies. After washing, the membranes were
incubated with HRP-conjugated anti-mouse IgG antibodies or anti-goat IgG antibodies (1: 1,000).
Detection was completed using enhanced chemiluminescence (ECL, Amersham Pharmacia).1
Immunohistochemistry assays were conducted as previously described (Hsieh et al.,
Proteomics 2006). In brief, tissue specimens for examination by light microscope were fixed in 40
g/L neutral buffered formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin.
For immunohistochemical, we used mouse anti-human ERBB3 as primary antibodies (1:500
dilution in PBS abcam, Cambridgeshire, UK). Second antibodies used were an HRP-conjugated
rabbit anti-mouse IgG. HRP activity was finally detected using diaminobenzidine tetrahydrochloride
(DAB) as substrate for 3 mins in accordance with the manufacturer’s instruction (Vecgtor
Laboratories, Burlingame, CA).
Migration and invasion assays
Invading and migration activities of HCC cells were analyzed using Boyden chambers (8µM pore
size, Corning Inc.), which were coated with Matrigel (BD Biosciences) (Matrigel:DMEM = 1:3,
Becton Dickinson Labware, Bedford, MA, USA) for invasion assays but not for migration assays,
and the bottom chambers were supplemented with specified amounts of fetal bovine serum or
recombinant NRG1. After being serum starved for 24 hours, hepatoma cells were detached with
trypsin, washed with PBS, re-suspended in serum free media and 200µl cell suspension (2×105
cells/ml) was added to the upper chamber in the each well of a 24-well Transwell plate and cultured
for 16 h or 40 h. Cells on the upper side of the membranes were removed using a cotton swab, and
cells on the underside were fixed in chilled 100% methanol for 60 min and stained with Geimsa
staining reagent. The cells were counted in five independent microscopic fields at 20-fold
magnification. For migration with wound assays, cells were starved and wound was created with a
p-200 pipette tip 16 hours after seeding followed by maintained in the defined culture conditions.
For those with silencing of EGFR, HER2, and ERBB3, after wound creation, cells were maintained
in 2% FBS to minimize proliferation. Images were attained 0h, 24h and 48h after wound creation.
These experiments were performed twice in duplicate.
Cell proliferation assays
Colorimetric sodium 3´-[1- (phenylaminocarbonyl)- 3,4- tetrazolium]-bis (4-methoxy- 6-nitro)
benzene sulfonic acid hydrate (XTT) assays to measure cell proliferation were performed according
to the manufacturer’s instruction (Roche). Cells (3 × 103) were seeded in 96-well microplates,
cultured in the absence of serum for 24 h, and then treated with or without NRG1 (1 ng/ml) for
additional 24, 48, or 72 h followed by XTT assays, respectively.
1.
Hsieh SY, Shih TC, Yeh CY, Lin CJ, Chou YY.Lee YS. Comparative proteomic studies on
the pathogenesis of human ulcerative colitis. Proteomics 2006;6:5322-5331.