Supplementary figure legends
Supplementary Figure S1. The cytotoxic effect of IB on HNSCC cells, and the overall impact of IB on
tumor growth. (a) The MTT assay for IB in 4 HNSCC cell lines (OECM-1, SAS, CAL-27, FaDu) and a
primary HNSCC culture. n=3 for each concentration. (b) The representative results of in vivo fluorescent
imaging of mice implanted with SAS cells with or without IB 2 mg/kg twice a week treatment. (c)
Quantification of the bioluminescent signals of the SAS-bearing mice with or without IB treatment. n=5 for
each group.
Supplementary Figure S2. IB reverses EMT. (a) Western blot of Twist1, the epithelial marker γ-catenin, and
the mesenchymal marker N-cadherin in the primary HNSCC cells receiving different concentrations of IB
treatment for 24 h. -actin was a loading control. (b) Western blot of Twist1, the epithelial marker E-cadherin,
and the mesenchymal marker N-cadherin in SAS cells receiving 1μM IB treatment for 24 h. -actin was a
loading control.
Supplementary Figure S3. IB does not affect TWIST1 mRNA expression in HNSCC cells. Quantitative RTPCR of TWIST1 was performed in OECM-1 and SAS cell lines and the primary HNSCC culture receiving
treatment of IB (1μM) or control for 24 h. n=3 for each group. Data represent mean ± S.E.M.
Supplementary Figure S4. IB regulates the stability of Twist1 and the expression of other EMT
regulators. (a)-(b) Cycloheximide (CHX) pulse-chase assay. (a) Upper: western blot of Twist1 in FaDu-Twist1
cells receiving 1μM IB or the control vehicle treatment. CHX 50μg/ml was added as indicated time points for
inhibiting the de novo protein synthesis. Lower: relative optical density of Twist1 at different time points. (b)
Upper: western blot of NEDD9 in 293T cells transfected with NEDD9 expression vector receiving 1μM IB or
the control vehicle treatment. CHX 50μg/ml was added as indicated time points for inhibiting the de novo
1
protein synthesis. Lower: relative optical density of NEDD9 at different time points. (c) Western blot of the
EMT regulators (Zeb1, Zeb2, Twist1, Snail, and Slug) in OECM-1 cells, SAS cells, and the primary HNSCC
culture with or without 1μM IB treatment for 24 h. -actin was a loading control.
2
Supplementary Table S1. IC50 of IB in HNSCC cells
Cell line
Tissue origin
OECM-1
SAS
CAL-27
FaDu
Primary HNSCC culture
Human head and neck squamous cell carcinoma
Human head and neck squamous cell carcinoma
Human head and neck squamous cell carcinoma
Human head and neck squamous cell carcinoma
Human head and neck squamous cell carcinoma primary culture
3
IC50 of IB
(μM)
7.24
5.08
3.95
2.78
5.70
Supplementary Table S2. Information for antibodies used in the experiments
Protein
B23
Bmi1
Assay
WB
PLA
-actin
WB
-Trcp
WB
CD44
FC
c-Myc
WB
DOCK3
E-cadherin
WB
WB; IF
FBXL14
WB; PLA
GAPDH
WB
HA tag
MMP9
WB
WB
N-cadherin
WB
NEDD9
WB
P65
-catenin
WB
WB
Slug
WB
Snail
Twist1
Vimentin
WB
WB; IP;
IHC; PLA
WB; IF
Zeb1
WB
Zeb2
WB
Antibody
MAB4500, Millipore
#05637, Upstate
Biotechnology, Inc.
A1978, Sigma-Aldrich
Corp.
LS-C39733, Lifespan
Biosciences, Inc.
#3516, Cell Signaling
Technology, Inc.
#OP30L, Calbiochem,
Inc.
ab75911, Abcam, Inc.
#4065, Cell Signaling
Technology, Inc.
LS-C166071, Lifespan
Biosciences, Inc.
#2118, Cell Signaling
Technology, Inc.
#05-904, Millipore
sc-21733, Santa Cruz
Biotechnology, Inc.
#610921, BD
Biosciences
sc-33658, Santa Cruz
Biotechnology, Inc
#1546-1, Epictomics
sc-8415, Santa Cruz
Biotechnology, Inc
#9589S, Cell Signaling
Technology, Inc.
Ab 85931, Abcam, Inc.
sc-81417, Santa Cruz
Origin
mmab
mmab
Dilution Incubation period
1/5000 overnight, 4C
1/1000 overnight, 4C
mmab
1/10000 1 hour, 4C
mmab
1/1000
overnight, 4°C
mmab
1/200
20 min, 4C
mmab
1/500
overnight, 4C
rpab
rpab
1/500
1/1000
overnight, 4C
overnight, 4C
rpab
1/500
overnight, 4°C
rmab
1/3000
overnight, 4°C
mmab
mmab
1/1000
1/2000
overnight, 4°C
overnight, 4°C
mmab
1/1000
overnight, 4C
mmab
1/500
overnight, 4C
rmab
mmab
1/1000
1/1000
overnight, 4°C
overnight, 4C
mmab
1/1000
overnight, 4C
rpab
mmab
1/1000
1/200
overnight, 4C
overnight, 4°C
V6630, Sigma-Aldrich
Corp.
sc-25388, Santa Cruz
Biotechnology, Inc
ab25837, Abcam, Inc.
mmab
1/1000
overnight, 4C
rpab
1/1000
overnight, 4C
rpab
1/1000
overnight, 4C
Abbreviations: PLA, Proximity ligation assay; FC, flow cytometry; IF, immunofluorescence; IHC,
immunohistochemistry; IP, immunoprecipitation; mmab, mouse monoclonal antibody; rmab, rabbit monoclonal
antibody; rpab, rabbit polyclonal antibody; WB, western blot
4
Supplementary Table S3. Experimental details for 2.5D culture
Experiments
Time-lapse
microscopy for
tracking cell
movement
Type of culture plate
3.5 cm dish (Corning
#430165, 8 cm2)
Immunofluorescence Lab-Tek® II chamber
and confocal
slide (#154461, 4 cm2 )
microscopic
examination
Gel volume
1 mL
Cell number
1 X 105
Medium volume
1 mL
0.5 mL
5 X 104
0.5 mL
5
Supplementary Materials and Methods
Culturing HNSCC cells on top of a thick layer of collagen (2.5D culture). For preparation of cells culturing
on top of a thick layer of collagen (2.5D culture), we cultured the cells on plastic dishes to 50% confluency.
Then we harvested the cells by trypsinizing them with 0.1% Trypsin-EDTA under 37ºC for 5 min, suspended
the cells to a concentration of 0.5 x 106/ml, and confirmed that the suspended cells were single cells by
microscopic examination. The 1.7mg/ml collagen solution (3ml) was prepared by mixing 1.7ml of 3mg/ml
PureCor bovine collagen solution (Advance Biomatrix, Inc., San Diego, CA), 0.6ml of 5x medium, 20 μl of
1M NaOH, and added water to a total volume of 3ml. The collagen solution was allowed by polymerize in the
tissue culture incubator at 37ºC for 1hr. Finally, we plated the cells to the thick collagen-coated dishes, then
added proper amount of serum-containing culture medium with 1μM IB or a control vehicle. The experimental
details about the size of the dish, the gel volume, the cell number, and the volume of the medium are shown in
Supplementary Table S3.
Aanalysis of cellular morphology in 2.5D culture. For analysis of cellular morphology, the area and the
perimeter of the cells were defined by drawing around the cell shape in phase contrast images and determined
by MetaMorph® software (Molecular Devices, Inc., CA). The morphology index was calculated as the
perimeter2/4π area. For a round cell, the ratio is 1, and an elongated cell has an elevated index.
Quantification of motility speed by time-lapse microscopy. To quantify the speed of cells in 2.5D system, we
tracked the moving pathways of individual cells for 8 hrs in a humidified, CO2-equilibrated chamber with a
Zeiss LSM700 Confocal Microscope (Carl Zeiss AG, Oberkochen, Germany). The cell motility speed was
calculated by MetaMorph® software in a randomly selected high power field and presented as microns per
minute. The experimental detail of the 2.5D culture for measuring motility speed is shown in Supplementary
Table S3.
6
Immunofluorescent staining and 3D image reconstruction. For immunofluorescent staining of F-actin in
2.5D culture, we coated each well of the Lab-Tek® II chamber slide (#154461) with 0.5 ml of the 1.7 mg/ml
collagen solution, then dispensed 5x104 cells on collagen, covered with 0.5 ml medium and allowed to adhere
for 16 hr. After incubation, the cells were fixed, permeabilized, and stained with Alexa-488 coupled to
phalloidin (Invitrogen Corporation, CA). Images were captured by laser confocal microscopy (Olympus
FV1000, Olympus Corporation, Japan). For 3D image reconstruction of cells, sequential Z sections were
obtained by confocal microscopy, and images were reconstructed by Olympus FV10-ASW 1.7 software. DAPI
was used for nuclear staining. The antibodies used in immunofluorescent staining are listed in Supplementary
Table S2. The experimental detail of the 2.5D culture for immunofluorescence is shown in Supplementary Table
S3.
3D invasion assay. Cells were plated for attachment, the supernatant was removed, and the cells were covered
with the 1.7 mg/ml collagen solution. Medium containing 15% FBS was added as a chemoattractant, and the
plates were incubated at 37C, 5% CO2 for 24 h. After incubation, the cells were fixed and stained with Alexa488 coupled to phalloidin. Confocal Z slices were collected each well at 50 µm from the bottom of the well with
a laser confocal microscope, and images of sequential Z sections were obtained and reconstructed by Olympus
FV10-ASW 1.7 software. The invasion index was calculated as the number of cells between 30 to 50μm divided
by the total number of cells, and was corrected by the cell viability with or without IB treatment. The data are
presented as the percentage of the invasion index of the control clone.
Flow cytometry. For analysis of CD44 expression, cells were treated with IB for 24 h and then were
resuspended in PBS and incubated with FITC-conjugated anti-CD44 antibody and evaluated with a
FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The antibodies used in flow cytometric assay are
listed in Supplementary Table S2.
7
Spheroid formation assay. For spheroid formation assay, 1 x 104 cells treated with 1μM IB or a control vehicle
for 24 h were suspended in defined serum-free medium composed of serum-free DMEM/F-12 (Gibco-BRL,
Gaithersburg, MD), N2 supplement, 10 ng/mL human recombinant bFGF and 10 ng/mL EGF (all from R&D
Systems Inc., Minneapolis, MN). The number of spheroids larger than 50m was counted after 14 days.
Soft agar colony formation assay. For soft agar colony formation assay, 1 x104 cells treated with 1μM IB or a
control vehicle for 24 h were suspended in culture medium containing 0.3 % agar (Sigma Chemical, St Louis,
MO). The agar-cell mixture was plated on top of a bottom layer with 0.5 % agar-medium mixture. After 14 days,
the viable colonies were counted.
In vivo fluorescent imaging. The mice were injected with luciferin intraperitoneally 10-15 minutes before
imaging. Mice were anesthetized by 2% isoflurane and the IVIS-50 system (Caliper Co., Hopkinton, MA, USA)
was used for image acquisition. Regions of interest were drawn over the signals, and the total flux of fluorescent
signals was expressed as photons per second. For monitoring the development of tumor growth, imaging was
performed weekly.
8