Download SUPPLEMENTARY MATERIALS AND METHODS

SUPPLEMENTARY MATERIALS AND METHODS
Identification of Foxq1, Twist1 and Zeb2 binding sites To identify Foxq1, Twist1 and
Zeb2 binding sites in the regulatory regions of target genes, we obtained the most recent
annotated versions of the 10-20 kb genomic sequences upstream and downstream of the
transcription start site of the Twist1, Zeb2 and PDGFRs locus in human and mouse from
the UCSC Genome Browser (http://genome.ucsc.edu/). Three transcription factor binding
site
prediction
tools,
rVista
(http://rvista.dcode.org),
CONTRA
v2
(http://www.dmbr.ugent.be/prx/bioit2-public/contrav2/index.php) and EpiTect ChIP tool
(http://www.sabiosciences.com/chipqpcrsearch.php?app=TFBS) were run to identify
putative transcription factor binding sites. The Consensus binding sequences for all three
transcription factors were sown in Supplementary Table S4.
ChIP-PCR assay ChIP was carried out using Magna ChIPTM G kit (Millipore) according
to the manufacturer’s instructions. Briefly, HMLE/Foxq1, HMLE/Twist1 or HMLE/Zeb2
cells were grown to 80% confluence and cross-linked at room temperature with 1%
formaldehyde. The cells were then scraped from the plates and lysed with cell lysis buffer.
After centrifugation, the cell lysates were resuspended in nuclear lysis buffer and sonicated
at 4°C. The sonicated lysate was diluted 10-fold in ChIP dilution buffer, and
immunoprecipitated with anti-V5 or control mouse IgG antibodies (Invitrogen) overnight at
4°C. The immunoprecipitated complexes were reverse cross-linked for DNA isolation. A
quantitative PCR (qPCR) analysis was performed using the Fast SYBR Green Master Mix
(Applied Biosystem). The experiments were performed in triplicate and repeated three
times. Primer sequences are shown in Supplementary Table S5.
Luciferase reporter assay HEK293T cells (1.5×105 cells per well in 24-well plates) were
co-transfected with 0.6 μg pcDNA3.1–Foxq1, pcDNA3.1–Twist1, pcDNA3.1-Zeb2 or an
empty control vector and 200 ng of the Firefly luciferase reporter genes along with 5 ng
pRL–TK plasmid (Promega). Firefly luciferase activity was normalized to the
corresponding Renilla luciferase activity. The experiments were performed in triplicate and
repeated two times.
Data mining for correlation of gene expressions:
We downloaded level 3 gene
expression (RNAseV2) data from The Cancer Genome Atlas (TCGA) database by
accessing the website (https://tcga-data.nci.nih.gov/tcga/findArchives.htm. The data were
first log-transformed and normalized, and then corrected for batch effects such as tissue
source site and center etc. For correlation between gene expressions, we calculated the
Spearman correlation coefficients and p values after normalization of the datasets for preprocessing. The survival curve was calculated using the Kaplan-Meier estimate and the
difference between the groups was tested using a log-rank test. A difference of P < 0.05
(two-sided) was considered statistically significant. No correction for multiple testing was
attempted due to the nature of this study.
Flow cytometry analysis Cells were harvested with trypsin and washed with PBS
containing 2% FBS and 2% BSA. Antibodies against CD44 (FITC, #555478, BD
Pharmingen), CD24 (PE, #555428, BD Pharmingen) were added per the manufacturer’s
instructions and incubated on ice for 30 minutes. Samples were then analyzed using a
FACScan flow cytometer (BD Biosciences).
Immunoblotting and immunofluorescence Immunoblotting and immunofluorescence
were performed with the following antibodies: anti-V5 (Invitrogen), total and phosphorPDGFRα and β (Cell Signaling), E- and N-cadherin (BD Transduction Laboratories), α-, βand -catenin (BD Transduction Laboratories), fibronectin (BD Transduction Laboratories),
vimentin (Fisher Scientific), and β-actin (Santa Cruz Biotechnology Inc.).
Cell proliferation assay For the cell proliferation assay, cells were seeded in triplicate at
the density of 2.5 × 103 per well in 96-well plates on day 0. Cell proliferation was measured
with an MTT assay kit (Fisher Scientific, Pittsburgh, PA) on day 1, 4, 7 and 10. For the
drug treatment, the cells were treated with different doses for 0, 12, 24, 48 and 72 hrs and
cell cell proliferation was measured by MTT assay. All these experiments were repeated
two times.
Cell proliferation assay was also used to study chmoresistance. For this purpose, the cells
were treated with chemotherapy agents (Dox and Pac) for 24 hours with different doses as
indicated. After being cultured in a drug-free growth medium for another 48 hours, the
surviving cells were quantified by MTT assay. The IC50 is determined using compuSyn
software.
Transwell migration assay and Boyden chamber invasion assay Cell migration and
invasion assays were performed using the 24-well control chamber and 24-well Matrigel
invasion chamber according to the manufacturer’s instructions (BD Biosciences, San Jose,
CA). Different cells were seeded in a density of 2.5 × 104 /chamber with DMEM medium.
DMEM medium with 10% FBS was used as a chemoattractant. About 48 hours after
seeding, migrating and invading cells were fixed and stained with a HAME-3 stain set
(Fisher Scientifics). The experiments were performed in triplicate and repeated two times.
Clonogenic assay For clonogenic assays, cells were seeded into a 48-well plate (1 × 104
cells/well). After 24 hours, the cells were treated with various concentrations of paclitaxel
or doxorubicin (Sigma) for an additional 24 hours. After culturing in a drug-free culture
medium for another 48 hours, an aliquot of the harvested cell population was seeded onto
a 10 cm dish. Crystal violet staining was used to count the colonies after a 10-day culture
in the medium. The experiments were performed in duplicate and repeated three times.
Tumor xenograft studies
1) To measure the effect of Foxq1 on drug resistance, 4T1 and 4T1/Foxq1-cells (2 × 104
cells/50µl) were injected subcutaneously into one flank of female BALB/c mice. Mice
with palpable tumors (about 6 days after cell implanting) were treated with vehicle
control (DMSO) or paclitaxel (4 mg/kg) thrice weekly by intraperitoneal (IP) injection.
The experiment was terminated and the mice were euthanized on day 25. Tumor size
was measured with calipers every other day. Tumor volume (V) was determined by the
equation V = (L × W2) × 0.5, where L is the length and W is the width of the tumor.
2) To study the effect of Foxq1 in tumorigenesis, we performed an in vivo limiting
dilution assay. Different numbers (2 × 104, 9 × 103, 3 × 103) of 4T1 non-targeted
control cells (4T1/NT) and Foxq1 knockdown cells (4T1/ sh3) were injected into the
No. 4 fat pad of BALB/C female mice. The injection volume is 50 µl. The tumor
incidence was assessed in 30 days.
3) To study the role of PDGFR in tumorigenesis and metastasis, HMLER-Foxq1 and its
derived PDGFRα and β individual or double knockdown cells (1.5 × 106 cells/0.2 ml in
a 50:50 solution of PBS and Matrigel) were injected into the fat pads of female NCR
nu/nu mice, respectively. The mice were euthanized 90 days after cell implanting.
Tumors and lungs were removed and embedded into paraffin for immunohistochemical
staining.
4) To study the effect of imatinib treatment on chemosensitivity in vivo, HMLER-Foxq1
cells (2 × 106 cells/0.2 ml in a 50:50 solution of PBS and matrigel) were orthotopically
injected into the mammary fat pads of female NCR nu/nu mice and these mice were
randomly assigned into four different groups. Three weeks after cells implantation,
these mice received the following treatments thrice weekly by intraperitoneal (IP)
injection: vehicle control (PBS), imatinib (10 mg/kg), doxorubicin (5 mg/kg), or
imatinib plus doxorubicin, respectively. After 70 days of cell injection, mice were
euthanized and tumors were removed and measured.
In a similar study, 4T1 cells were injected into BALB/c mice as previously described
and six days after cell implanting, these mice were also randomly assigned into four
treatment groups and received the following treatment through IP injection: vehicle
control (PBS plus DMSO), imatinib (20 mg/kg), paclitaxel (4 mg/kg), or imatinib plus
paclitaxel. Vehicle control and imatinib were delivered daily and paclitaxel was
delivered every other day. Mice were euthanized at day 24 and tumors were removed
and measured.
H&E staining and immunohistochemistry assay (IHC)
For H&E staining, paraffin-embedded sample slides were de-paraffinized, hydrated and
then stained with hematoxylin for one minute. After rinsing, the slides were stained with
eosin for one minute, rinsed and sealed with cover slips using Permount. For IHC, mouse
monoclonal anti-p27 antibody (1:200, Vector Laboratory), rabbit polyclonal anti-BAX
antibody (1:200, Zymed Labs, Inc.), and rabbit polyclonal anti-cleaved caspase 8 antibody
(1:50, Cell Signaling Technology). These antibodies were used in conjunction with
ImmunoCruz goat ABC staining kit (Santa Cruz Biotechnology, INC.).