Supplementary Methods
Generation of NKX6.3 stable cells. We generated stable NKX6.3 transfectants of
AGS and MKN1 cells, AGSNKX6.3 and MKN1NKX6.3, stably expressing human
NKX6.3, as well as mock transfectants, AGSMock and MKN1Mock cells, as
described previously.1 Stable expression of NKX6.3 was confirmed in AGSNKX6.3
and MKN1NKX6.3 cells by western blot analysis.
For NKX6.3 and GKN1 silencing, shNKX6.3 and shGKN1 were cloned
into the pcDNA3.1 expression vector and pSilencer 3.1 H1-neo (Invitrogen,
Carlsbad, CA, USA). AGSNKX6.3 and MKN1NKX6.3 cells were transfected in 60
mm-diameter dishes with the expression plasmids (2 μg total DNA) using
Lipofectamine
Plus
transfection
reagent
(Invitrogen)
according
to
the
manufacturer's recommendations.
For effects of BMP4 signaling pathway on intestinal metaplasia, AGSMock
and MKN1Mock, AGSNKX6.3 and MKN1NKX6.3 cells were incubated with BMP4 (10
and 50 ng/ml) from 24 and 48 hours and used for western blot and
immunoprecipitation analysis.
Expression of differentiation-related genes. After quantification of mRNA
extracted from non-cancerous gastric mucosae, cDNA was synthesized using the
reverse transcription kit from Roche Molecular System (Roche, Mannheim,
Germany), according to the manufacturer's protocol. For QPCR, 50 ng cDNA was
amplified using Fullvelocity SYBR Green QPCR Master Mix (Stratagene, La
Jolla, CA, USA) and 20 pmol/μl of each primer (forward and reverse) using
Stratagene Mx 3000P QPCR system, according techniques previously published.2
NKX6.3 and CDX2 mRNAs were quantified by SYBR Green Q-PCR and
normalized to mRNA of the glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). Sequences of the primers are described in Supplementary Table 1.
Data are reported as relative quantities according to an internal calibrator using
the 2-△△CT method.3 All samples were tested in duplicate, and the mean values
were used for quantification. The mean value of mRNA expression in gastric
mucosae without H. pylori infection, intestinal metaplasia and atrophy was used
as a control. mRNA expression change in each case was further normalized to
mean value of those in control gastric mucosae and reduced mRNA expression
was defined less than 0.5 fold change.
To investigate whether ablation of NKX6.3 is associated with intestinal
metaplasia, the expression of CDX2, Muc2, SOX2, and Muc5ac mRNA were also
analyzed using real-time PCR in AGSMock, MKN1Mock, AGSNKX6.3 , and
MKN1NKX6.3 cells. In addition, the effects of NKX6.3 silencing with shNKX6.3 on
the expression of gastric and intestinal markers were also analyzed. Real-time
PCR was performed using SYBR Green Q-PCR Master Mix (Stratagene, La Jolla,
CA, USA), according to the manufacturer's instructions. Each mRNA was
quantified by SYBR Green QPCR and normalized to mRNA of the housekeeping
gene, GAPDH. The primer sequences are described in Supplementary Table 1.
Next, expression of CDX2 and SOX2 proteins was examined in NKX6.3 stable
cells before and after NKX6.3 silencing with shNKX6.3.
Chromatin immunoprecipitation. For assessing the NKX6.3 binding activity in
the promoter region of CDX2, SOX2 and TWSG1, chromatin immunoprecipitation
assays were performed using the Thermo Scientific Pierce Agarose chromatin
immunoprecipitation kit (Thermo Scientific Pierce, Rockford, IL, USA), as
previously described.1 Briefly, AGSMock, MKN1Mock, AGSNKX6.3 and MKN1NKX6.3
cells were cultured in a 10-cm dish for 4 days. The cells were fixed with 1%
formaldehyde in PBS for 10 min, washed twice with ice-cold PBS and resuspended in lysis buffer. Nuclei were recovered by centrifugation and MNase
digestion was carried out at 37°C for 15 min. Nuclei were lysed and the extracts
were immunoprecipitated with 4 µg of antibody against NKX6.3 at 4°C overnight.
Normal rabbit IgG was used as the negative control. Protein-bound DNA was
recovered using affinity chromatography purification columns according to the
manufacturer’s protocol (Thermo Scientific Pierce), and 5 µl of lysed nuclei were
also purified under the same procedure and used as input. DNA amplification was
performed by PCR using primers for the CDX2, SOX2 and TWSG1 promoter
described in Supplementary Table 1. Amplification products were separated on a 2%
agarose gel.
Effect of H. pylori CagA and GKN1 on intestinal metaplasia. To determine the
effect CagA and/or GKN1 on intestinal metaplasia, the genes associated with
intestinal metaplasia were examined in AGSMock, AGSNKX6.3, MKN1Mock, and
MKN1NKX6.3 cells at time course after transfection with CagA and shGKN1 as
described previously.4 The primer sequences are shown in Supplementary Table 1.
The expression of CagA and differentiation-associated proteins was examined by
western blot as previously described.5
Immunofluorescent study. Rabbit anti-NKX6.3 (Abcam, Cambridge, MA, USA)
and SOX2 (Cell Signalling, Beverly, MA, USA) polyclonal antibody, and mouse
anti-GKN1 (Abcam), Muc5ac, Muc2 (Leica, Bannockburn, IL, USA), and CDX2
(Biogenex, San Ramon, CA, USA) monoclonal antibody (Abcam) and alexa-488
conjugated goat anti-rabbit IgG, texas-red conjugated goat anti-mouse and -rabbit
IgG (Invitrogen) secondary antibodies were used to visualize immunoreactivity.
The specificity of the reaction was tested by incubation with non-immune mouse
serum (Invitrogen).
Co-immunoprecipitation. AGSMock, AGSNKX6.3, MKN1Mock and MKN1NKX6.3
cells were treated with BMP4 and washed with PBS and lysed at 4°C with PBS,
pH 7.2 containing 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10mM
NaF, 1.0mM NaVO4, and 1.0% protease inhibitor cocktail (Sigma, St. Louis, MO,
USA) as previously described.6 Equal protein aliquots (1.0mg) were
immunoprecipitated with 2.0 μg of antibodies to BMP4 (Cell Signaling) plus
protein A/G-agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as
described manufacture’s recommendation. Immunoprecipitated proteins were
resolved on 12% SDS-polyacrylamide gels and transferred to PVDF membranes
(BioRad, Richmond, CA, USA). The membranes were blocked for 1 hr with PBS
containing 0.1% Tween 20 (PBS-T) and 5% non-fat dry milk (Sigma) and reacted
with antibodies against BMPR-II, BMP4 (Cell Signaling) or TWSG1 (Abcam) at
each diluted 1:1,000. The membranes were washed with PBS-T, incubated for 1 hr
at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG
antibody (Sigma) diluted 1:5,000 and developed with ECL plus Western blotting
detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
Immunoreactive bands were identified by co-migration of prestained protein size
markers (Fermentas, Glen Burnie, MD, USA). To confirm equivalent protein
loading and transfer, the blots were stripped and re-probed for GAPDH (Santa
Cruz Biotechnology).
Supplementary reference
1. Yoon, J.H., et al. Gastrokine 1 induces senescence and apoptosis through
regulating telomere length in gastric cancer. Oncotarget 5, 11695-11708 (2014).
2. Yoon, J.H., et al. Inactivation of the Gastrokine 1 gene in gastric adenomas and
carcinomas. J Pathol 223, 618-625 (2011).
3. Pfaffl, M.W. A new mathematical model for relative quantification in real-time
RT-PCR. Nucleic Acids Res 29, e45 (2001).
4. Yoon, J.H., et al. Gastrokine 1 inhibits the carcinogenic potentials of
Helicobacter pylori CagA. Carcinogenesis 35, 2619-2629 (2014).
5. Choi, W.S., et al. Gastrokine 1 expression in the human gastric mucosa is
closely associated with the degree of gastritis and DNA methylation. J. Gastric
Cancer 13, 232-241 (2013).
6. Guang, W., et al. MUC1 cell surface mucin attenuates epithelial inflammation
in response to a common mucosal pathogen. J Biol Chem 285, 20547–20557
(2010).
Supplementary figure 1. NKX6.3 functions as a transcriptional factor for
CDX2 and SOX2 genes. (A, B) A putative promoter activity was characterized
between -6 kb and +1 kb relative to transcription start site of CDX2 and SOX2
genes by chromatin immunoprecipitation-PCR. The binding activity of NKX6.3
was detected in all fragments. (C, D) NKX6.3 occupancies were 7.5- and 8.4-fold
enriched at the CDX2-transcription start site-5376~5215 and SOX2-transcription start
site-4051~3838 compared to control.
Supplementary figure 2. NKX6.3 controls gastric differentiation by GKN1
independent manner. (A) Stable NKX6.3 transfectants increased GKN1 mRNA
expression in a time dependent manner, whereas GKN1 silencing with shGKN1
significantly down-regulated GKN1 mRNA expression. (B) In both NKX6.3
stable transfectants, GKN1 silencing with shGKN1 did not affect NKX6.3 mRNA
expression. (C, D) GKN1 silencing did not affect the effect of NKX6.3 on SOX2
and CDX2 mRNA expression.
Supplementary Table 1. Primer sequences used in this study
Primer name
Sequences
For Real-time RT-PCR
NKX6.3
for DNA copy number
GAPDH
for DNA copy number
NKX6.3
for mRNA expression
CDX2
SOX2
Muc5ac
Muc2
GKN1
GAPDH
for mRNA expression
F: 5’- AACCCCACACTTCCTTCCTC -3’
R: 5’- TCTCCCGCACAGACTTACCT -3’
F: 5’- ACCCAGAAGACTGTGGATGG -3’
R: 5’- TTCTAGACGGCAGGTCAGGT -3’
F: 5’- TCTTTCTGCTTCTGGGGTGT -3’
R: 5’- GTCCAGCGGCTTGTTGTACT -3’
F: 5’- GCGGAACCTGTGCGAGTG -3’
R: 5’- GACTGTAGTGAAACTCCTTCTCCAGC -3’
F: 5’- TTGCTGCCTCTTTAAGACTAGGA -3’
R: 5’- CTGGGGCTCAAACTTCTCTC -3’
F: 5’- GCTCAGCTGTTCTCTGGATGAG -3’
R: 5’- TTACTGGAAAGGCCCAAGCA -3’
F: 5’- CTTCGACGGACTCTACTACAGC -3’
R: 5’- CTTTGGTGTTGTTGCCAAAC -3’
F: 5’- CAAAGTCGATGACCTGAGCA -3’
R: 5’- CTTGCCTCTTGCATCTCCTC -3’
F: 5’- AAATCAAGTGGGGCGATGCTG -3’
R: 5’- GCAGAGATGATGACCCTTTTG -3’
For shRNA
shNKX6.3
shGKN1
5’- GGACCGCGCACCCTCGGAGAACGAGGACG -3’
5’- GCCCAAACAAAGTCGATGAC -3’
For chromatin immunoprecipitation analysis
CDX2-4533~-4356
CDX2-4376~-4215
CDX2-3899~-3796
CDX2-552~-395
CDX2-9~+141
SOX2-4425~-4246
SOX2-3972~-3790
SOX2-3731~-3545
SOX2-3051~-2838
SOX2-2659~-2432
SOX2-2388~-2215
SOX2-2165~-2007
TWSG1-2165~-1916
TWSG1-1758~-1463
F: 5’- CCCTTTCTCCTGAGGCTCTT -3’
R: 5’- AGGCTGCAGGATAATGGGAT -3’
F: 5’- ATCCCATTATCCTGCAGCCT -3’
R: 5’- GAAGCAGATGTCTCCTTGCC -3’
F: 5’- TCAGTATAGATGGCCAAGCTGA -3’
R: 5’- ACTCTTACCTTGCATTGACCTG -3’
F: 5’- AGTCAGAGATGCAGCCTGAA -3’
R: 5’- CCTAAGAGATGGGCCATTCC -3’
F: 5’- CCTCGACGTCTCCAACCATT -3’
R: 5’- CCCACCTCCTTCCCACTAG -3’
F: 5’- TCTCACCAAATTGTACCAGCA -3’
R: 5’- AAGTGACCATAGCAGCAATCA -3’
F: 5’- TGTCAAATAGGGCCCTTTTCAG -3’
R: 5’- GGAACCTGCAGAGTCTTGGA -3’
F: 5’- CAGATGCAGAAAAGAAGCGTT -3’
R: 5’- GTCTCCTCAGCACTCCTCAA -3’
F: 5’- TGTTCCTGCTATCACCATCTTC -3’
R: 5’- GGGGTTGGCTAGGTCTTTTATT -3’
F: 5’- ACCACCCTTATCCACACCAAT -3’
R: 5’- CTCGAGGTAAGTGGGAGGTT -3’
F: 5’- ATGATTTGGTTCCGCAGCTT -3’
R: 5’- CCCTGCTGGTAGATTCGCTT -3’
F: 5’- GCCTTCTCTTCCCCAGATCT -3’
R: 5’- CGGAAAGGGTGAGATTGGGT -3’
F: 5’- ATTAATTATTCAAAAACTATGCAGTACTTT -3’
R: 5’- AAACTGTTGCAGTATCATGTATTATGTA -3’
F: 5’- AAATAATGAATATGTAAAAAATTGTTTTAATTT -3’
R: 5’- AAAATCATCCTTGAAAATTATCCTAGAG -3’