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Page 4 of 14 Tafur et al. J Cancer Metastasis Treat 2018;5:xx I http://dx.doi.org/10.20517/2394-4722.2018.102

Unless otherwise noted, this medium was also supplemented with physiological levels of: glucose (5 mmol/L;
(Gibco A24940), sodium pyruvate (0.1 mmol/L; Gibco 11360-070), Na-lactate (1 mmol/L; Sigma L7022) and
L-glutamine (0.5 mmol/L; Sigma G7513. This same medium, but without phenol red (powder, R9010-01; US
Biological, Swampscott, MA, was used in assays where cells were treated with β-Estradiol or Tamoxifen.

MCT1 inhibitor SR 13800, β-Estradiol and Tamoxifen (Tocris Bioscience, Bristol, UK) solutions were used at
concentrations of 100 nmol/L, 10 nmol/L and 1 µmol/L, respectively.

Imaging of 3-dimensional structures
Cells were cultured in 3-dimensional Matrigel and glass bottom 24-well plate (MatTek Corporation, MA) for
seven days. Phase contrast images of the 3-dimensional structures of epithelial and mesenchymal cells were
imaged using a Zeiss Axio Observer inverted microscope (Carl Zeiss MicroImaging Inc., Thomwood, NY)
equipped a QImaging Retiga EXi CCD digital camera (QImaging, Surrey, BC). For immunofluorescence
images cell lines grown in 3-dimensional Matrigel at the seventh day were fixed in 2% formaldehyde in 1× PBS
for 20 min. Cells were washed with 1× PBS and then treated with 0.1% Triton-X 100 for 10 min. Cell were washed
with 1× PBS and blocked with 1% bovine serum albumin (BSA) for 20 min. After blocking, cells were
incubated with F-actin-staining solution for 20 min at room temperature. F-actin-staining solution was
prepared with 5 µL Alexa-488 Phallotoxin (Invitrogen, Carlsbad, CA) stock in 200 µL 1X PBS for each well to
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be stained. SYTOX orange dye (Invitrogen, Carlsbad, CA) was used to stain nuclei. Images were captured
using a Zeiss Pascal confocal system with a 40× 1.2 NA objective (Carl Zeiss Microscopy, Narashige, MN).

Transient transfection of siRNA
MCF-7 cells were seeded in 250 uL at a density of 150, 000 cells/well in twenty-four-well plates coated with 200 µL
of the mix of 1:1 Matrigel /Physiological modified medium supplemented with 17 µmol/L Insulin-Transferrin-
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Selenium solution (Gibco, Grand Island, NY) and transiently transfected with Trilencer-27 human siRNA
(siNT and siGATA3 “A”) at a final concentration of 10 nmol/L (Origene, Rockville, MD) with Lipofectamine
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RNAiMAX transfection reagent (Thermo Scientific, Rockford, IL). After 8 h of transfection cells were
overlaid with 250 µL of physiological modified medium containing 4% Matrigel . Culture medium was
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replaced with fresh medium every day and cells were used for each assay after 72 or 96 h after transfection.

Growth and viability assays
MCF-7 cells were seeded at a density of 150, 000 cells/well and recovered from Matrigel by incubating them
with 5 mM EDTA (Gibco, Grand Island, NY) in ice cold 1X PBS for 30 minutes at 4 °C and centrifuged for
5 min at 1000 rpm. Recovered cells were digested with 0.25% trypsin for 4 min and resuspended in DMEM
with 10% FBS. The cell number was calculated on hemocytometer at specific time point. Viability assays
were performed three times for each cell group.

Real-time qPCR and primers
Total RNA was isolated from cell lines using TRIzol Reagent (Ambion RNA, Carlsbad, CA), following
the manufacturer’s instruction. cDNA was synthesized 1 μg of total RNA using iSCRIPT cDNA synthesis
kit (Bio-Rad, Hercules, CA). SYBR green based SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA) was
utilized to perform real-time PCR from 50 ng of cDNA. Gene expression was normalized to TATA-box
binding protein using 2 −ΔCt method. Specificity of the primer sets was confirmed by melting curve analysis.

cDNA arrays
Real-time qPCR was performed in two TissueScan breast cancer and normal tissue cDNA arrays I and II
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(Origene, Rockville, MD). Gene expression was normalized to normal tissue using 2 − ΔCt method.