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Page 4 of 12 Park et al. J Cancer Metastasis Treat 2019;5:17 I http://dx.doi.org/10.20517/2394-4722.2018.84

Tumor data selection
We selected data from The Cancer Genome Atlas (TCGA) project to represent the breast invasive carcinoma
(BRCA). We downloaded publicly available 1,222 RNA-Seq data of 1,092 breast cancer cases from TCGA
database. Using FPKM values from all 1,222 samples, the expression level of AF1q was compared to that of
ICAM-1.

RT and real-time PCR analysis
Total RNA was subjected to reverse transcription (RT) with a High capacity RNA-to-cDNA kit (Thermo
Fisher Scientific), and the resulting cDNA was subjected to real-time polymerase chain reaction (RT-qPCR)
analysis with BrightGreen qPCR master mix (ABM) and a StepOne real time PCR system (Thermo Fisher
Scientific). The amplification protocol comprised 40 cycles of incubations at 95 °C for 30 s and at 60 °C for
60 s. PCR primer sequences (forward and reverse) were as follows: 5’-TGAGTACAGCACCTTCAACTTC-3’
and 5’-GGGAAAGGAGTGGAAAGGAAG-3’ for AF1q; 5’-CAATGTGCTATTCAAACTGCCC-3’ and
5’-CAGCGTAGGGTAAGGTTCTTG-3’ for ICAM-1; 5’-CAGAGGGCTACAATGTGATGGC-3’ and
5’-GCTGAGGATTTGGAAAGGGTG-3’ for HPRT1.

Plasmid construction
Full-length AF1q cDNA was inserted into pLUTdNB, which is a pTRIPZ base modified doxycycline-
inducible vector by cloning the HindIII and XhoI fragments as previously described. Short hairpin RNA
(shRNA) of AF1q was purchased from Open Biosystems. Empty pLUTdNB, and pTRIPZ-scramble were
used as controls for pLUTdNB-AF1q, and pTRIPZ-AF1q shRNA, respectively.

Viral production and infection
Lentivirus was produced by co-transfection of HEK 293T cells (ATCC) with the lentiviral constructs
pVSV-G and psPAX2 (Addgene). Transfections were carried out using Lipofectamine 2000 (Invitrogen). For
enforced AF1q expression or shRNA targeting, cells underwent lentiviral transduction and were selected
[15]
using 1 μg/mL puromycin (Thermo Fisher Scientific) as previously described . After antibiotic selection,
cells were cultured in complete medium including 1 µg/mL doxycycline (Thermo Fisher Scientific) for AF1q
or shRNA induction.

Western blot
For blots of whole-cell lysates, cells were lysed directly in GLB buffer (2% SDS, 10% glycerol and 50 mmol/L
Tris, pH 6.8), boiled, and separated by electrophoresis on a 4%-12% SDS-PAGE gradient gel. Proteins were
transferred to PVDF membrane (Millipore) and blocked in 5% skim milk (Bio-Rad) in 0.05% PBST. Rabbit
monoclonal anti-AF1q, ICAM-1, and β-actin antibodies were purchased from Abcam. After the appropriate
antibody incubations, an enhanced chemiluminescence (Denville) system was used for developing blots.

Reporter gene construct, transfection, and Luciferase assay
Dual-Luciferase reporter assays were performed using an ICAM-1 reporter plasmid. To construct reporter
plasmids, 850 bases of an ICAM-1 promoter fragment were cloned to between the XhoI and HindIII sites of the
4
pGL4-Luciferase reporter plasmid (Promega). Briefly, 1 × 10 cells were plated in each well of a 96-well plate. The
next day, the reporter plasmid (100 ng) and Renilla plasmid (10 ng, Promega) were co-transfected into the cells
with lipofectamine 2000 (Thermo Fisher Scientific). After 48 h, cells were washed with PBS and assayed with
the Dual-Luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Luciferase
activity was determined using Synergy H1 Multi-Mode reader (Biotek). Quantitation of luminescent signal from
reporter plasmid was normalized by quantitation of the luminescent signal from Renilla.

Surface and intracellular staining
Cells were harvested using non-enzymatic dissociation solution (Corning) and washed once with PBS. For
staining of ICAM-1, cells were incubated in Human Fc Block (BD) for 10 min. Thereafter, cells were stained