SUPPLEMENTARY APPENDIX
SUPPLEMENTARY MATERIALS AND METHODS
Families
NMTC frequency in the sixty families was as follows: one family had 5 members affected, five had 4
members affected, twelve had 3 members affected, and the remaining forty two families had 2 affected
members. In the 145 patients, the most common tumor was the classic variant of PTC (cPTC) (n = 106,
73.6%), except for three probands, whom had FTC.
Establishment of PCCL3 cells clones
Transfections of PCCL3 cells were performed in 35 mm plates (Nunclon, Oakville, Ontario), using
3.5x105 cells, 3 g of plasmid, and 7.5 l of Lipofectamine (Lipofectamine 2000, Invitrogen TM, Life
Technologies, NY, USA). Clones of PCCL3 cells stably expressing different plasmids were established
after selection with 500 μg/ml geneticin (G418, Sigma-Aldrich, Oakville, Ontario), for two weeks. The
clones were selected using cloning cylinders with 8 mm x 8 mm (Merck Millipore, USA). Geneticin was
used at a final concentration of 500 μg/ml for the maintenance of the PCCL3 clones.
Establishment of TPC-1 cells pools
Transfections of TPC-1 cells were performed in 35 mm plates (Nunclon), using 3.5x105 cells, 2 g of
plasmid, and 2 l of Lipofectamine (Life Technologies). Pools of TPC-1 cells stably expressing different
plasmids were established after selection with 1000 μg/ml G418 (Sigma-Aldrich), for two weeks.
Geneticin was used at a final concentration of 500 μg/ml for the maintenance of the TPC-1 cells pools.
Western blot
Protein lysates from PCDNA3, 16 WT and 16 MUT TPC-1 pools were boiled for 5 min in Laemmli
Buffer
and
resolved
in
10%
SDS-PAGE
mini
gels.
Proteins
were
transferred
onto
polyvinylidenedifluoride membranes (Bio-Rad) using a Trans-Blot cell system (Bio-Rad). Membranes
were probed using the appropriate antibodies, and specific binding was detected via a secondary
peroxidase-conjugated antibody (Bio-Rad), followed by a chemiluminescence assay. The antibodies used
in this study were rabbit anti-FOXE1 (1:500) from Abcam (ab138310) (Cambridge, USA), mouse anti-
1
ACTB (1:1000) from Sigma-Aldrich as an endogenous control to ensure equal loading of cell protein per
lane, anti-rabbit IgG peroxidase conjugated (1:3000), and anti-mouse IgG peroxidase conjugated (1:3000)
(Thermo Scientific, Brookfield, USA). Membranes were analyzed in the ChemiDoc system (Bio-Rad)
with the Image Lab™ software. For densitometric analysis, images from experiment were digitalized and
analysed using ImageJ software (National Institutes of Health).
Quantitative real-time PCR (qRT-PCR)
RNA was extracted from the PCCL3 clones and TPC-1 pools, using the RNeasy Mini Kit (QIAGEN,
Hilden, Germany). Reverse transcription of 1 or 2 µg of RNA was performed, using 200 U of SuperScript
II RT Reverse Transcriptase (InvitrogenTM), and random primers (InvitrogenTM), according to the
manufacturer's instructions.
The synthesized cDNA from pools was used for quantitative real time PCR (qRT-PCR) to determine
the relative expression of the WNT5A gene in the three pools of TPC-1 cells. RT-PCR amplifications were
performed using pre-developed primers and probe (Inventoried TaqMan Gene Expression Assays ID;
hs00998537_m1; Applied Biosystems), and TaqMan Universal PCR Master Mix (Applied Biosystems),
according to the manufacturer’s protocol. The endogenous control used was Actin beta (ACTB)
(Inventoried TaqMan Gene Expression Assays ID: Hs01060665_g1; Applied Biosystems). Four different
concentrations of a standard sample of cDNA of TPC-1 cells (100, 25, 6.25, 0.78 ng) were used to apply
the relative standard curve method. The cDNA of TPC-1 cells was used as calibrator, and samples
expressions were normalised to PCDNA3 pool expression. All reactions were performed in triplicate.
The cDNA from the PCCL3 clones was used to determine the relative expression of human FOXE1
(hFOXE1). The endogenous control used was ACTB. To apply the relative standard curve method for
ACTB, five concentrations of a standard sample of cDNA from PCCL3 cells (10; 5; 2; 0.4; 0.08 ng) were
used. In the construction of standard curve for hFOXE1 we used seven different concentrations of the
same standard sample (10; 2; 0.4; 0.08; 0.016; 0.0032; 0.00064 ng). RT-PCR amplifications were
performed using SYBR Green, dNTPs, AmpliTaq Gold DNA (Applied Biosystems) and 10 pmol/μL
primer forward e reverse (InvitrogenTM). All reactions were performed in triplicate. Primers sequences are
indicated in Supplementary Table 1.
Real-time PCR was performed in 96-well reaction plates (MicroAmp Optical 96-well Reaction Plate,
Applied Biosystems), on an ABI PRISM 7900 Sequence Detection System (Applied Biosystems).
2
SUPPLEMENTARY RESULTS
Establishment of PCCL3 clones with stable expression of wild type and p.A248G FOXE1
The integration of the different plasmids in the PCCL3 clones was confirmed by sequencing the
human FOXE1 gene in cDNA samples from each cell population (details of primers and PCR conditions
are indicated in Supplementary Table 1). Sequencing analysis showed that, as expected, PCDNA3 clone
cells did not display human FOXE1 expression. Sequencing results also confirmed the stable integration
of wild type human FOXE1 gene (with 16 alanine residues) in the 16 WT clone, and the stable integration
of human FOXE1 gene with the variant under study p.A248G (and with 16 alanine residues) in the 16
MUT clone (data not shown).
Establishment of TPC-1 pools with stable expression of wild type and p.A248G FOXE1
The integration and expression of the different plasmids in the TPC-1 pools, were confirmed by
sequencing the FOXE1 gene in cDNA samples from each cell population. The sequencing analysis
revealed that, as expected, the PCDNA3 pool exhibited only expression of the endogenous FOXE1 gene.
In these cells, the endogenous FOXE1 gene had the wild type sequence, and 14 alanine residues in
homozygosity. The 16 WT pool showed expression of the stably integrated wild type FOXE1 gene (with
16 alanine residues) and a low (endogenous) expression of the wild type FOXE1 gene (with 14 alanine
residues). The 16 MUT pool showed expression of the stably integrated FOXE1 gene expression with the
variant under study p.A248G (and with 16 alanine residues), and a low (endogenous) expression of wild
type FOXE1 gene (with 14 alanine residues) (data not shown).
SUPPLEMENTARY FIGURES
a
b
Supplementary Figure 1 – DNA sequence analysis of exon of the FOXE1 gene in a normal control (A)
and in the proband of family 25 (B), showed a C to G transversion (c.743C>G), which is expected to lead
3
to the replacement of an alanine (GCC) residue by a glycine (GGC) residue at codon 248 (p.A248G). The
arrow indicates the identified variant.
a
b
FOXE1
β-ACTIN
Supplementary Figure 2 – Expression of the FOXE1 protein. (A) Representative Western blot showing
levels of FOXE1 and β-actin in HeLa cells (positive control), TPC-1 pools (PCDNA3, 16 WT and 16
MUT). β-Actin was used as a loading control. (B) Quantitative results of FOXE1 protein expression in
HeLa cells (positive control), PCDNA3, 16 WT and 16 MUT pools.
Supplementary Figure 3: Effect of the FOXE1 p.A248G variant on thyroid cell growth. Proliferation
assays were performed for the three PCCL3 clones (PCDNA3, 16 WT and 16 MUT) by direct cell
counting at 48, 96 and 144 hours (a). Relative proliferation of 16 WT and 16 MUT were compared with
the PCDNA3 (control) at all time-points, and statistically significant differences between 16 MUT and
4
both 16 WT and PCDNA3 clones were only observed at 144 hours (b). Error bars indicate the standard
deviation. Values of proliferation assays correspond to an average of eight replicates in three independent
experiments, and data were analyzed by non-parametric Kruskal-Wallis test. P values <0.05 were
Product
FOXE1 Locus
Primer Nameª
Sequence (5’-3’)
size
(bp)
MgCl2
(mM)
Annealing
Temperature
(ºC)
considered statistically significant.
Supplementary Figure 4 - Effect of the FOXE1 p.A248G variant on cell migration. Cell migration of the
three PCCL3 clones (PCDNA3, 16 WT and 16 MUT) was tested by wound healing assays, at time-points
3, 6, 9, 24, 27, 30 and 54 hours (a). Migration rates of the 16 WT and 16 MUT were calculated in relation
to PCDNA3 (control), at all time-points, and statistically significant differences between 16 MUT and
both 16 WT and PCDNA3 clones were only observed at 27 hours (b), 30 and 54 hours (data not shown).
Error bars indicate the standard deviation. Values of migration assays correspond to an average of seven
replicates in three independent experiments, and data were analyzed by non-parametric Kruskal-Wallis
test. P values <0.05 were considered statistically significant.
SUPPLEMENTARY TABLE
Supplementary Table 1 – Oligonucleotide primer pairs and conditions for PCR amplification and
sequencing analysis of the FOXE1 gene
5
FOXE1 promotor
FOXE1 exon 1
qRT-PCRc
FOXE1-Prom-F
GTGGAGAGGACCAGCCTCA
FOXE1-Prom-R
CTTCTGCGGAGAGCTCAGG
FOXE1-1F
CTGAGCTCTCCGCAGAAGG
FOXE1-1R
GTCAAGCGCCCAGTAGTTG
FOXE1-2F
CCTCACACTCAACGACTGCTT
FOXE1-2R
AGCGGCAAAGATCGCACT
FOXE1-3F
GCCAATGGCGCGGAAAACA
FOXE1-3R
CCTCTCTCCATTCCTGTTGC
hFOXE1-F
GACCACGGTGGACTTCTAC
hFOXE1-R
GGACACGAACCGATCTATC
a
Primers used for PCR amplification and sequencing.
DMSO 6% (v/v).
c
Primers used in the qRT-PCR for human FOXE1.
b
6
527
1.6
60
499
1.6b
61
586
1.6b
60
439
1.6b
58
157
1.6b
58