Supplementary Material and Methods Materials and Methods RNA extraction, reverse transcription (RT) and real-time PCR. Total RNA from cultured cells was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) as the manufacturer instructed. cdnas were amplified and quantified in ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) using dye SYBR Green I (Molecular Probes, Invitrogen, Carlsbad, CA). Expression data were normalized to the geometric mean of housekeeping gene GAPDH to control the variability in expression levels and calculated as 2 -[(C t of gene) (C t of GAPDH)], where C t represents the threshold cycle for each transcript. Primers and Oligonucleotides. Real-time PCR primer: AKIP1, forward: 5 -CCAACCCTT AGTGCTTCC-3 and reverse: 5 -TCGACTCGCCTCTGTGATA-3 ; VEGFC, forward: 5 -CGGACTCGACCTCTCGG-3 and reverse: 5 -TGGACACAGACCGTAACTGC-3 ; ANGPT1, forward: 5 -ACCGGATTTCTCTTCCCAGA-3 and reverse: 5 -CCGACTTCA TGTTTTCCACA-3 ; BCL2L1, forward: 5 -TTCAGTGACCTGACATCCCA-3 and reverse: 5 -CTGCTGCATTGTTCCCATAG-3 ; BCL3, forward: 5 -CCTATACCCCATGATGTGCC -3 and reverse: 5 -GCACCACAGCAATATGGAGA-3 ; BMP2, forward: 5 -CACTGTGCG CAGCTTCC-3 and reverse: 5 -CCTCCGTGGGGATAGAACTT-3 ; CCL1, forward: 5 -ATGCAGATCATCACCAC AGC-3 and reverse: 5 -AAGCAACATCTGGAG AAGGG -3 ; CCL3, forward: 5 -GGCTCTCTGCAACCAGTTCT-3 and reverse: 5 -TGAAATTCTG TGGAATCTGCC-3 ; CCL5, forward: 5 -TACACCAGTGGCAAGTGCTC-3 and reverse: 5 -TGTACTCCCGAACCCATTTC-3 ; CCL19, forward: 5 -CCTGCTGGTTCTCTGGAC TT-3 and reverse: 5 -CTCACGATGTACCCAGGGAT-3 ; CCND1, forward: 5 -TCCTCTC CAAAATGCCAGAG -3 and reverse: 5 -GGCGGATTGGAAATGAACTT -3 ; CXCL1, forward: 5 -GAAAGCTTGCCTCAATCCTG-3 and reverse: 5 -CTTCCTCCTCCCTTCTGG
TC-3 ; CXCL3, forward: 5 -ATCCCCCATGGTTCAGA AA-3 and reverse: 5 -ACCCTGC AGGAAGTGTCAAT-3 ; CXLC6, forward: 5 -TTTGTCT GGACCCGGAAG-3 and reverse: 5 -GGCAATTTTATGATGCATGG-3 ; EDN1, forward: 5 -TCTCTGCTGTTTGTGGCTTG -3 and reverse: 5 -GACTGGGAGTGGGTTTCTCC-3 ; ENG, forward: 5 -TCCATGTCCTC TTCCTGGAG-3 and reverse: 5 -CTGAGGACCAGAAGCACCTC-3 ; ERBB2, forward: 5 -CTCCTCCTCGCCCTCTTG-3 and reverse: 5 -AGCATGTCCAGGTGGGTCT-3 ; FGF8, forward: 5 -CAGGTCCTGGCCAACAAG-3 and reverse: 5 -CTCTGCTTCCAAAGGTGT CC-3 ; GATA3, forward: 5 -AAAATGAACGGACAGAACCG-3 and reverse: 5 -TCTGA CAGTTCGCACAGGAC-3 ; HIF1A, forward: 5 -GAAGACATCGCGGGGAC-3 and reverse: 5 -TGGCTGCATCTC GAGACTTT -3 ; IGFBP1, forward: 5 -CTGCGTGCAGGA GTCTGA-3 and reverse: 5 -TCCTCCTCAGTTATCTCCGTG-3 ; IL1A, forward: 5 -ACTGCCCAAGATGAAGACCA-3 and reverse: 5 -CCGTGAGTTTCCCAGAAGAA-3 ; IL1B, forward: 5 -GAAGCTGATGGCCCTA AACA-3 and reverse: 5 -AAGCCCTTGCTG TAGTGGTG-3 ; IL6, forward: 5 -AGTGAGGAACAAGCCAGAGC-3 and reverse: 5 -GTC AGGGGTGGTTATTGCAT-3 ; IL8, forward: 5 -TCCTGATTTCTGCAGCTCTGT-3 and reverse: 5 -AAATTTGGGGTGGAAAGGTT-3 ; JUNB, forward: 5 -GAACAGCCCTTCT ACCACGA-3 and reverse: 5 -AGGCTCGGTTTCAGGAGTTT-3 ; KLK3, forward: 5 -GCCTGAGACAACAAATGGGT-3 and reverse: 5 -CTGAATGAAGAGTCTGGGGC-3 ; KRT3, forward: 5 -GAGCGGGAACAGATCAAGAC-3 and reverse: 5 -TCCACTTGGTCT CCAGGACT-3 ; MMP9, forward: 5 -ACGACGTCTTCCAGTACCGA-3 and reverse: 5 -TTGGTCCACCTGGTTCAACT-3 ; SOX9, forward: 5 -GACGCTGGGCAAGCTCT-3 and reverse: 5 -GTAATCCGGGTGGTCCTTCT-3 ; THBS1, forward: 5 -CAATGCCACA GTTCCTGATG-3 and reverse: 5 -CACAGCTCGTAGAACAGGAGG-3 ; TNF, forward: 5 -CTGCTGCACTTTGGAGTGAT-3 and reverse: 5 -AGATGATCTGACTGCCTGGG-3 ; TNFSF13B, forward: 5 -CTCAAGACTGCTTGCAACTGA-3 and reverse: 5 -TTCTAGGG
CACTTCCCCTTT-3 ; GAPDH: 5 -ATTCCACCCATGGCAAA TTC-3 and 5 -TGGGATTTCCATTGATGACAAG-3. Primers used for human AKIP1 promoter reporter construction: P1-luc, forward: 5 -GCC GCTAGCGAGCGTGTCCCTCAAAGT and reverse: 5 -GCCAGATCTCCCCTTCCATTTT CATCTAC; P2-luc, forward: 5 -GCCGCTAGCAATGTAGACCAAGCAGAAAGAG and reverse: 5 -GCCAGATCTCCCCTTCCATTTTCATCTAC; P3-luc, forward: 5 -GCCGCTAG CCCCGAGTACCTGGGACTACA and reverse: 5 -GCCAGATCTCCCCTTCCATTTTCA TCTAC; P4-luc, forward: 5 -GCCGCTAGCGAGCGTGTCCCTCA AAGT and reverse: 5 -GCCAGATCTGCCTCTTTCTGCTTGGTCTA; P4-luc-mut, forward: 5 -ATGTGAATCC GTACTTCCCACTGCTCGGGAGAGAGAGCTGCGCTG and reverse: 5 -CAGCGCAGC TCTCTCTCCCGAGCAGTGGGAAGTACGGATTCACAT; P4-luc-del, forward: 5 -AATATGTGAATCCGTACTTCCCACTGGGGAGAGAGAGCTGCGCTGAGGGATT ATG and reverse: 5 -CATAATCCCTCAGCGCAGCTCTCTCTCCCCAGTGGGAAGTAC GGATTCACATATT. AKIP1 promoter primer used for ChIP assay: Region 1, forward: 5 -TGGAGTCAGAC CCCAAGA-3 and reverse: 5 -GAACAGCTCGGGATTGAG-3 ; Region 2, forward: 5 -AA T GTAGACCAAGCAGAAAGAG-3 and reverse: 5 -GAGGTTGCAGTGAGACGAG-3 ; Region 3, forward: 5 -CCCGAGTACCTGGGACTACA-3 and reverse: 5 -GCTG CTTAA CTTGGTGGGTAT-3 ; Region 4, forward: 5 -CCTGC CTTTATACCCACC-3 and reverse: 5 -CATCTGTCCACGCTGTCA-3 ; Region 5, forward: 5 -GCAGTTAACCAGGCAACTC G-3 and reverse: 5 -CCCTTCCATTTTCATCTACCC-3. GAPDH, forward: 5 -GGTAGGG AGTTCGAGACCAG-3 and reverse: 5 -TCAACGCAGTTCAGTTAGGC-3. AKIP1 sirna#1: CCUUCAGAACAAUGGCUGAAU (within exon3); AKIP1 sirna#2: GUCAGUGUGGGAAAUAUUAUUCA (within exon3).
Supplementary Figure Legends Supplementary Figure 1. Western blotting analysis of Nkx2-8 expression in Kyse30 (A) and Kyse510 (B) ESCC cells stably expressing Nkx2-8-cDNA and Nkx2-8-shRNA(s). α-tubulin was used as a loading control. Supplementary Figure 2. Downregulation of Nkx2-8 promotes ESCC angiogenesis in vivo. (A) Xenograft model in nude mice (n = 6/group). Representative images of the tumor-bearing mice. The indicated cells were injected into the dorsal flank of the mice. (B) Tumor volumes were measured on the indicated days. (C) Mean tumor weights. (D) Quantification of MVD in the tumors formed by control cells and Nkx2-8 silenced cells as indicated by the expression of CD31. (E) Expression of VEGF-C mrna in the tumors formed by vector cells, Nkx2-8-transduced cells, scrambled-vector cells and Nkx2-8-silenced cells. Each bar represents the mean ± SD of three independent experiments. * P < 0.05. Supplementary Figure 3. Downregulation of Nkx2-8 promotes NF-κB activity. EMSA of endogenous NF-κB activity in randomly selected 4 Kyse30/vector tumors and Kyse30/Nkx2-8 tumors (left panel), or 4 Kyse30/RNAi-vector tumors and Kyse30/Nkx2-8 RNAi-#2 tumors (right panel). OCT-1 DNA-binding complex served as a control. Each bar represents the mean ± SD of three independent experiments. * P < 0.05. Supplementary Figure 4. Expression of Nkx2-8 mrna in human ESCC. Real time-pcr analysis of Nkx2-8 mrna expression in 10 freshly collected paired primary ESCC tissues (T) and the adjacent non-cancerous tissues (ANT) from the same patients. Expression levels were normalized to GAPDH. Each bar represents the mean ± SD of three independent experiments. * P < 0.05
Supplementary Tables Supplementary Table 1. Clinicopathological characteristics of studied patients and expression of Nkx2-8 in ESCC Factor NO. (%) Gender Male 239 73.8 Female 85 26.2 Age (years) 57 158 48.8 >57 166 51.2 Clinical stage I 41 12.6 II 122 37.7 III 102 31.5 IV 59 18.2 T classification T 1 42 13.0 T 2 69 21.3 T 3 201 62.0 T 4 12 3.7 N classification N 0 139 42.9 N 1 183 56.5 N 2 2 0.6 N 3 0 0 M classification No 300 92.6 Yes 24 7.4
Histological Well 99 30.6 Moderate 127 39.2 Poor 98 30.2 Vital status Alive 115 35.5 Dead 209 64.5 Expression of Nkx2-8 Low expression 175 54.0 High expression 149 46.0
Supplementary Table 2. Correlation between the clinicopathological features and expression of Nkx2-8 Patient characteristics Nkx2-8 expression Low or none High P-value Gender Age (years) Clinical stage T classification N classification M classification Histological differentiation Vital status Male 132 107 Female 43 42 57 84 74 >57 91 75 I 2 39 II 64 58 III 61 41 IV 48 11 T 1 9 33 T 2 42 27 T 3 116 85 T 4 8 4 N 0 66 73 N 1 109 74 N 2 0 2 N 3 0 0 No 155 145 Yes 20 4 Well 48 51 Moderate 63 64 Poor 64 34 Alive 41 74 Dead 134 75 0.527 0.824 <0.001 <0.001 0.030 0.003 0.027 <0.001
Supplementary Table 3. Univariate and multivariate analysis of different prognostic parameters in patients with ESCC by Cox-regression analysis Univariate analysis Multivariate analysis No. patients P Regression coefficient (SE) P Relative risk 95% confidence interval N classification N 0 139 N 1 183 <0.001 0.141 <0.001 1.772 1.337-2.348 N 2 2 N 3 0 Expression of Nkx2-8 Low expression 175 High expression 149 <0.001 0.145 <0.001 0.447 0.336-0.594