Ghosh S et al. Supplementary Materials
Contents
- Supplementary TableS1
- Supplementary TableS2
- Supplementary TableS3
- Supplementary TableS4
- Supplementary TableS5
- Supplementary TableS6
- Legends to Supplementary Figures
- Supplementary FigureS1
- Supplementary FigureS2
1
TableS1. Oligonucleotide primers used in the study.
Primers / Location / Analytical purpose / Sense / Antisense / Size (bp)SST / Intron 2 / Control for deletion / 5’-GCTCAAATGTTTCTGCAACC-3’ / 5’-CTTTGTAGCTCGTGATGTGA-3’ / 168
ROBO1 Exon 8 / Coding region of exon2 / HD / 5’-CATATCAACCACCACAGTCA-3’ / 5’-TCTGGCAGATGTAATAACCA-3’ / 105
ROBO2 Exon 2 / Coding region of exon2 / HD / 5’-CACGACTCTGAACTGCAAG-3’ / 5’-ACTCACTGCTTCACCAAGAT -3’ / 223
SST Exon 2 / Coding region of exon / control for HD / 5’-AACCAGACGGAGAATGATG-3’ / 5’-GAAAGCTAACAGGATGTGAAA-3’ / 176
ROBO1 meth / -252 to +91 bp # / Methylation / 5’-GATTTCCGATTCCGAGCAA-3’ / 5’-GCAGAGAAGGGCAGAGAGG-3’ / 343
ROBO2 meth / -688 to -407 bp # / Methylation / 5’-ACGTAGGAGTTCGGATTCTC-3’ / 5’-TCCTAGAAAGTGTTCCACAAA-3’ / 302
RARβ2 ( K2) / Coding region of exon 1 / DNA integrity control / 5’-AGAGTTTGATGGAGTTGGGT-3’ / 5’-CATTCGGTTTGGGTCAATCC-3’ / 229
β-3A ADAPTIN (K1) / Exon 1-intron1 / DNA digestion control / 5’-TGCCCTCTGGACTGGAACCT-3’ / 5’-CCTGAGCCCAGCCCAAGTC-3’ / 445
ROBO1/DUTT1 meth M / -257 to +60 bp / MSP / 5’-GTTTGGATTTTCGATTTCGA-3’ / 5’-ATAAAAAAAACGACCGCCG -3’ / 317
ROBO1/DUTT1unmethU / -256 to +60bp / MSP / 5’-TTTGGATTTTTGATTTTGA -3’ / 5’-ATAAAAAAAACAACCACCAC -3’ / 316
ROBO2 meth M / -770 to -580bp / MSP / 5’-CGGGGGTGTTTGTAGTTTC-3’ / 5’-ACGAACCTCTCCTTTACAACG-3’ / 190
ROBO2 unmeth U / -770 to -581bp / MSP / 5’-TGGGGGTGTTTGTAGTTTTG-3’ / 5’-CAAACCTCTCCTTTACAACAAT-3’ / 189
ROBO1 Exon 6-8 / Coding region of exon / mRNA expression / 5’-AAATATGGTGGGCAAAGCTG-3’ / 5’-CTGGATGACTGTGGTGGTTG-3’ / 216
ROBO2 Exon 8-9 / Coding region of exons / mRNA expression / 5’-GGGTTACTACATCTGCCAGGCTT-3’ / 5’-AGGTGGAGGTCTATCTGTCAAAACAT-3’ / 97
B2M / Coding region of exon / Control for expression / 5’-GTGCTCGCGCTACTCTCTCT-3’ / 5’-TCAATGTCGGATGGATGAAA-3’ / 143
# Distance with respect to transcriptional start site.
# Microsatellite makers used in this study were from Genome Database: Ensembl release 49.
# Primer was designed using Primer3Input 0.4.0 software.
1
TableS2. Association between deletion of ROBO1/DUTT1 and ROBO2
dysplasia / HNSCCROBO2 / ROBO2
DEL+ / DEL- / DEL+ / DEL-
ROBO1/DUTT1 / DEL+ / 14 / 16 / 27 / 36
DEL- / 4 / 38 / 4 / 49
p value / 0.0003 / 0.00002
D: Deletion; ‘+’: Positive; ‘-’: Negative
1
TableS3. Correlation between MSRA and MSP
ROBO1/DUTT1 / ROBO2914 / MSRA
MSP
1234 / MSRA
MSP
1552 / MSRA
MSP
2323 / MSRA
MSP
1319 / MSRA / Methylated CpG
MSP / Unmethylated CpG
1047 / MSRA
MSP
2030 / MSRA
MSP
7216 / MSRA
MSP
2937 / MSRA
MSP
4786 / MSRA
MSP
3922 / MSRA
MSP
2073 / MSRA
MSP
5219 / MSRA
MSP
Hep2 / MSRA
MSP
SCC084 / MSRA
MSP
% of methylation / MSRA / 47(7/15) / 27(4/15)
obtained / MSP / 47(7/15) / 27(4/15)
Significance level / P-value / 0.0002 / 0.0001
TableS4. Association between deletion with methylation of ROBO1/DUTT1 and ROBO2
Dysplastic lesions / HNSCC samplesROBO1/DUTT1 / ROBO1/DUTT1
M+ / M- / M+ / M-
ROBO1/DUTT1 / D+ / 7 / 23 / 13 / 48
D- / 5 / 17 / 5 / 45
p value / 0.9243 / 0.1076
Dysplastic lesions / HNSCC samples
ROBO2 / ROBO2
M+ / M- / M+ / M-
ROBO2 / D+ / 7 / 11 / 7 / 24
D- / 1 / 33 / 6 / 74
p value / 0.0006 / 0.0266
D: Deletion; M: Methylation
‘+’: Positive; ‘-’: Negative
1
TableS5. Association between molecular alterations of ROBO1/DUTT1 and ROBO2
dysplasia / HNSCCROBO2 / ROBO2
Alt+ / Alt- / Alt+ / Alt-
ROBO1/DUTT1 / Alt+ / 16 / 13 / 32 / 40
Alt- / 2 / 21 / 3 / 36
p value / 0.00045 / 0.00007
Alt: Overall molecular alterations
‘+’: Positive; ‘-’: Negative
TableS6.Correlation between ROBO1/DUTT1 and ROBO2 molecular alterations with mRNA and protein expression.
Sample No. / ROBO1/DUTT1 / ROBO2Genetic/epigenetic alterations / mRNA status / Protein expression / Genetic/epigenetic alterations / mRNA status / Protein expression
Dysplastic lesions
L127 / D+M+ / ND / Low / D+M- / ND / Medium
L139 / D+M- / ND / Low / D-M- / ND / High
L154 / D+M- / ND / Medium / D+M- / ND / Medium
L162 / D-M- / ND / Normal / D-M- / ND / High
L158 / D+M- / ND / Low / D+M- / ND / Low
HNSCC samples
#1004 / D-M- / Normal / Normal / D-M- / Normal / Normal
#5165 / D+M+ / ↓ / Low / D-M- / Normal / High
#6817 / D+M- / ↓ / Low / D+M+ / ↓ / Low
#7077 / D-M- / Normal / Normal / D-M- / Normal / Normal
#1108 / D+M- / ↓ / Medium / D-M- / Normal / Normal
#2884 / D-M- / Normal / Normal / D-M- / Normal / Normal
#6835 / D+M+ / ↓ / Low / D-M- / Normal / Normal
#872 / D+M+ / ↓ / Low / D+M- / ↓ / Medium
#5303 / D+M- / ↓ / Low / D-M- / Normal / Normal
#6814 / D-M- / Normal / Normal / D-M- / Normal / Normal
#797 / D+M- / ↓ / Low / D-M+ / ↓ / Low
#1049 / D+M- / ↓ / Low / D+M- / ↓ / Medium
#6907 / D+M- / ↓ / Low / D-M- / Normal / Normal
#315 / D-M- / Normal / Normal / D-M+ / ↓ / Low
#6392 / D+M+ / ↓ / Medium / D+M+ / ↓ / Medium
#5497 / D+M- / ↓ / Low / D+M+ / ↓ / Low
#1087 / D-M- / Normal / Normal / D-M- / Normal / Normal
#944 / D+M- / ↓ / Low / D+M- / ↓ / Medium
#756 / D+M- / ↓ / Low / D-M+ / ↓ / Medium
#5733 / D+M- / ↓ / Low / D-M- / Normal / Normal
D: Deletion; M: Methylation; ‘+’: Positive; ‘-’: Negative; ‘↓’: Reduced expression
Legends for Supplementary Figures:
FigureS1. 5-aza-dC effects on ROBO1/DUTT1 and ROBO2 protein expression in Hep2 and SCC084 cell lines. Western analysis of SCC084 and Hep2 cell lysates showed 2.6 and 1.2 foldoverexpression of ROBO1/DUTT1 and ROBO2 proteins respectively after treatment with 20µM 5-aza-dC for 120 h. In Hep2,ROBO1/DUTT1 and in SCC084 cells ROBO2 protein expression remained unchanged after treatment with 5-aza-dC for 120h (data not shown). α Tubulin (sc-8035, Santa cruz biotechnology, U.S.A) was a control for the amount of loaded protein (30 µg total protein/lane).
FigureS2.Kaplan-Meier 5-year survival probability curves with cumulative survival of HNSCC patients by molecular alteration status in ROBO1/DUTT1 loci. The smooth line represented survival probability without molecular alterations and the dotted line represented the same probability with molecular alterations. N, total number of HNSCC samples.
1