Manuscript
Hosoda et al.
Supplementary Figure Legend---665 words
Supplementary Figure 1. Chromosomal copy number alterations (CNA) in gastric
adenocarcinomas. The array-based comparative genomic hybridization (aCGH) data
was obtained using MCG Whole Genome Array-4500. (a) Overall frequency of CNA in
163 gastric adenocarcinomas. Horizontal line: 4177 BAC clones on the array, mapped in
the human reference genome (Build 36.3) and aligned from chromosomes 1 to 22 and X.
Vertical line: frequency of gains (green) and losses (red). The peak regions of frequent
chromosomal gain were the MYC-PVT1 locus at 8q24.2 (45.6% of cases), the terminal
region of 8q (51.5%), the ERBB2 locus at 17q12 (48.1%), the PLUNC locus at 20q11.21
(51.9%), the PREX1 locus at 20q13.13 (60.0%) and the BIRC7 locus at 20q13.33
(62.2%). Regions of frequent chromosomal loss included the APC-MCC locus at 5q22.2
(33.9% of cases), the TUSC3 locus at 8p22 (30.5%), the JMJD2C locus at 9p24.1
(35.0%), the MLLT3 locus (32.6%) and CDKN2A locus (40.2%) at 9p21.3, and the
BCL2 locus at 18q21.33 (32.5%). (b) Representative profiles of chromosome 6p21
gain/amplification in 27 cases of gastric adenocarcinomas. BAC clones used for aCGH
analysis are displayed alongside a physical map of chromosome 6p. Overall frequency
of copy number gain (tumor/normal signal ratio of ≥1.3) detected by each clone is
shown as a percentage. Loci whose signal ratios are 1.3-1.5 are indicated in light pink,
1
Manuscript
Hosoda et al.
those 1.5-2.5 in dark pink, and those exceeding 2.5 in yellow.
Supplementary Figure 2. Genomic copy number profiles of chromosomes 4, 5 and 6
in gastric cancer cell lines. Horizontal lines: 1197 BAC clones on the MCG Whole
Genome-4500 array aligned from chromosomes 4 to 6 according to the human reference
map (Build 36.3). Vertical lines: signal ratio of gastric cancer cell line DNA/reference
DNA (human male). We screened copy number changes at the 6p21 locus in 22 gastric
cancer cell lines and found six, HSC58, HSC60, KATOIII, MKN45, NUGC3 and
Okajima, that harbored increased copies of chromosome 6.
Supplementary Figure 3. In vivo tumorigenic activity of 6p21 genes. (a) Tumor
development in nude mice. HEK293 polyclonal cells (5x106 cells) overexpressing a
recombinant 6p21 gene with a V5-His epitope tag at the carboxyl-terminal end were
injected subcutaneously into nude mice. Within three months, all HEK293 cells
transfected with the six genes formed tumors in mice with frequencies ranging from
17% to 83%. Most of the tumors (14/17) developed between 4 and 6 weeks after
inoculation. (b) Recombinant protein expression in tumors. Tumors were harvested and
subjected to Western blot analysis with anti-V5-HRP antibody (Invitrogen). Almost all
2
Manuscript
Hosoda et al.
of the tumors expressed the recombinant protein of the expected size, except for one
(lane 14. GTPBP2-3). Lanes: 1. GLO1-1, 2. GLO1-2, 3. GLO1-4, 5. GLO1-5, 6.
C6orf64-1, 7. C6orf64-2, 8. C6orf64-3, 9. C6orf64-4, 10. C6orf130-1, 11. TBCC-1, 12.
GTPBP2-1, 13. GTPBP2-2, 14. GTPBP2-3, 15. GNMT-1, 16. GNMT-2, 17. GNMT-3.
Supplementary Figure 4. Sequence variations of the GLO1 gene in 72 gastric tumors
and 23 cell lines. Genomic DNA of 23 gastric cancer cell lines and 72 primary gastric
tumors were directly sequenced after PCR amplification using a BigDye Terminator
v3.1 Cycle Sequencing Kit (Applied Biosystems) and analyzed on an ABI prism 3100
Sequencer (Applied Biosystems). (a) Schematic representation of the GLO1 gene.
Exons 1 to 6 are illustrated by boxes (white box, non-coding region; black box, coding
region). Arrowheads show the PCR primers used for exon amplification. The positions
of single nucleotide polymorphism (SNPs) detected in this study are shown in the figure.
(b) Allele frequencies of 7 loci detected in this study. (c) PCR primers for genomic
amplification and sequencing primers for screening of GLO1 mutation.
Supplementary Figure 5. Metabolme analysis using the CE-TOFMS (capillary
electrophoresis-time-of-flight mass spectrometry) method detected lower levels of
3
Manuscript
Hosoda et al.
metabolites in glycolysis, the pentose phosphate pathway (PPP) and TCA cycle.
Metabolic pathway map is shown with the ratio of metabolite concentration in
GLO1-downregulated gastric cancer cell/its reference cell in the parenthesis (Left,
NUGC3-shGLO1/NUGC3-scr; Right, MKN28-shGLO1/MKN28-scr). Downwards
arrow indicates the ratio being ≤0.7. Statistical significance is shown by solid line
(significant) or a dotted line (not significant).
4