a KYSE27-CON KYSE27- shcon shcon sh b Human Mouse CD31 Relative MVD 3.5 3 2.5 2 1.5 1.5 *** *** c KYSE15 KYSE27 sirna (nm) 5 1 Id2 Id2 sirna 5 1 sirna (nm) 5 1 Id2 sirna 5 1 Id2 [h] (pg per ml) d 3 2 1 KYSE27- KYSE27- CON-shCON -shcon Fibronec n DAPI Merged (Fibronec n + DAPI) E-cadherin KYSE27- CON-shCON KYSE27- -shcon KYSE27- -sh KYSE27- -sh [m] (pg per ml) 25 2 15 1 5 *** ** KYSE27- KYSE27- CON-shCON -shcon KYSE27 KYSE27- -sh Id3 Id3 sirna 5 1 Id3 Id3 sirna 5 1 DAPI e g Relative proliferation 15 1 5 Id4 CM from PIGF FGF Control Id4 sirna 5 1 CON -25-25 -25 Id4 Relative proliferation 15 1 5 Id4 sirna 5 1 CON Proliferation of fibroblasts Control -25-25 -25 Merged (E-cadherin + DAPI) f Relative proliferation CM from h 15 1 5 Proliferation of fibroblasts Relative proliferation 15 1 5 CM from (ng per ml) 5 1 5 1-7 -7 p-akt -7-7 AKT -1-1 p-mdm2 p53 - - 55 55 Supplementary Figure 1. -induced secreted from ESCC cells increases -mediated tumor angiogenesis but had no effect on fibroblast proliferation. (a) Quantification of CD31-MVD in the tumors established with KYSE27-, KYSE27--sh or vector control (female 6-8 week old nude mice, n = 3 per group; scale bar, 1 µm). (b) Human and mouse concentrations were determined in the serum of mice bearing xenografts expressing KYS27--shCON, KYSE27--sh or vector control (female 6-8 week old nude mice, n = 3). (c) Western blots showing the effects of, Id2, Id3 and Id4 knockdown on by using sirnas. (d) Immunofluorescence showed positive staining for fibronectin but not E-cadherin in fibroblasts compared with ESCC cells (i.e. KYSE27). Cell nuclei were counterstained with DAPI (scale bar, 1 µm). (e) Western blot showing the effects of CM from -overexpressing ESCC cells on PIGF, FGF and. (f) MTT assay showed no significant difference in proliferation of fribroblasts fed with the indicated CM from ESCC cells over a 24 hour-period. (g) The proliferation of fibroblasts treated with (1 ng per ml) for 24 hours was quantified. (h) Western blot analysis showing the expression of p-akt, AKT, p-mdm2, p53 and in fibroblasts upon treatment. Three biological replicates were performed for in vitro assays. Bars, s.d.; **, P <.1; ***, P <.1 by Student s t-test.
a Relative proliferation 2 15 1 5 Ab ** * CM b Ab Ab HUVEC migration CM CM Relative proliferation 25 2 15 1 5 Ab ** * CM Relative migration 3 2 1 Ab ** ** CM Relative migration 4 3 2 1 Ab ** ** CM c Tube formation d Cancer cell invasion Ab CM Ab CM KYSE27 Ab CM Ab CM KYSE27 Relative tube formation 3 *** *** 25 2 15 1 5 CM Ab Relative tube formation 4 3 2 1 Ab *** *** CM Relative invasion KYSE27 3 ** * 25 2 15 1 5 Ab CM Relative invasion 3 2 1 Ab KYSE27 ** ** CM Supplementary Figure 2. Paracrine effects of -activated fibroblasts on and ESCC cells. (a) Cell proliferation of fed with CM from -induced fibroblasts in the presence or absence of antibody was determined by MTT assay. (b) Comparison of chemotactic effect of different CM from fibroblasts as indicated on the migration of (scale bar, 1 µm). (c) Representative images and quantification of tube formation of treated with different CM from fibroblasts as indicated (scale bar,1 µm). (d) Comparison of invasion of ESCC cells under chemotactic influence of different CM from fibroblasts (scale bar, 1 µm). Three biological replicates were performed for in vitro assays. Bars, s.d.; *, P <.5; **, P <.1; ***, P <.1 by Student s t-test.
a HUVEC migration Blank CM from co-culture of fibroblasts with ESCC cells KYSE15- CON-shCON KYSE15- -shcon KYSE15- -sh KYSE15- -shcon + Ab Relative migration 3 25 2 15 1 5 ** ** ** * *** 5 *** ****** Relative migration 4 3 2 1 CM from co-culture of fibroblasts with CM from co-culture of fibroblasts with b Blank KYSE15- CON-shCON Tube formation CM from co-culture of fibroblasts with ESCC cells KYSE15- -shcon KYSE15- -sh KYSE15- -shcon + Ab Relative tube formation 3 25 2 15 1 5 CM from co-culture of fibroblasts with *** ** ** *** Relative tube formation 25 2 15 1 5 CM from co-culture of fibroblasts with *** *** *** ** Supplementary Figure 3. Effects of fibroblasts co-cultured with ESCC cells on migration and tube-forming capability of. (a) CM from co-culture of fibroblasts and -overexpressing ESCC cells had the highest propensity to induce migration of, compared with ESCC cells expressing -sh or vector control (scale bar, 1 µm). (b) Treatment with CM from co-culture of fibroblasts and -overexpressing ESCC cells significantly increased the tube-formation activity of (scale bar, 1 µm). Three biological replicates were performed for in vitro assays. Bars, s.d.; *P, <.5; **P, <.1; ***P, <.1 by Student s t-test.
a in fibroblasts 2.5 2 1.5 1.5 n = 22 r =.35 P =.16.5 1 1.5 2 2.5 3 in ESCC tissue b Breast cancer Stromal Low Stromal High P =.22 n = 53 Months Stromal Recurrence No Yes Total Low 25 1 26 High 17 1 27 Total 42 11 53 P =.5 Supplementary Figure 4. Correlation analysis of in ESCC and fibroblast and clinical relevance of stromal in breast cancer. (a) Graph correlating expression of in esophageal tissue with that of in fibroblasts. Correlation was assessed using Pearson s rank correlation coefficient. (b) The association between tumor stromal expression and the survival rates of patients with breast cancer in GEO database (GSE 914) was analyzed by Kaplan-Meier analysis; statistical significance was calculated by log-rank test; the Table below showed that the expression of stromal was significantly correlated with recurrence (P =.5 by Fisher exact test).
a Bone marrow of nude mice with established s.c. tumor CON-shCON -shcon -sh ESCC tumor xenograft CON-shCON -shcon -sh + R1 R1+ GFP+ GFP+ Lung of nude mice with established s.c. tumor CON-shCON -shcon -sh + R1 GFP+ b Mice bearing xenografts of CD11B+ Mice bearing xenografts of F4/8+ CD11b 15 CON-shCON -shcon -sh 1 5 GFP+ GFP+ CON -sh F4/8 CON-shCON -shcon -sh GFP +/ CD11b + cells GFP +/ F4/8 + cells 2 15 1 5 GFP+ CON -sh Mice bearing xenografts of CD335+ CD335 CON-shCON -shcon -sh GFP+ GFP +/ CD335 + cells 2 15 1 5 CON -sh Mice bearing xenografts of R2+ R2 CON-shCON -shcon -sh GFP+ GFP +/ R2 + cells 15 1 5 CON -sh Mice bearing xenografts of Gr-1 CON -sh 2 Gr-1+ GFP +/ Gr-1 + cells 15 1 5 GFP + CON -sh Mice bearing xenografts of Tie-2+ Tie-2 CON -sh GFP +/ Tie-2 + cells 2 15 1 5 GFP + CON -sh Supplementary Figure 5. Flow cytometric analysis of GFP + cells in bone marrow and lungs of nude mice bearing ESCC tumor xenografts. (a) GFP + /R1 + cell populations in the bone marrow, lungs and tumor of groups of mice with subcutaneous xenograft of ESCC cells expressing, -sh or control vectors (female 6-8 week old nude mice, n = 3 per group). (b) No significant difference was found in bone marrow CD11b +, F4/8 +, CD335 +, R2 +, Gr-1 + and Tie-2 + cell populations among the three groups (female 6-8 week old nude mice, n = 3 per group).
a Unsorted BM cells R1 - BM cells R1 + BM cells b Proliferation of R1 + BM cells 2.5%.32 % 95.1 % Relative proliferation 2 1.5 1.5 CM from fibroblasts treated with: * * Blank + Ab c Relative migration 8 6 4 2 CM from co-culture of fibroblasts with: Migration of R1 + BM cells *** *** *** ** Supplementary Figure 6. Paracrine effects of IGF-activated fibroblasts on migration and proliferation of R1 + bone marrow cells. (a) Post-sort analysis of R1 + bone marrow cells. (b) Comparison of proliferation of sorted R1 + bone marrow cells treated with the CM from -induced fibroblasts, in the presence or absence of antibody. (c) The migration of R1 + BMDCs attracted by different CM as indicated was determined. Three biological replicates were performed for in vitro assays. Bars, s.d.; *, P <.5; **, P <.1; ***, P <.1 by Student s t-test.
Tumor Mice bearing xenograft of KYSE15-CON -shcon KYSE15- -shcon KYSE15- -sh Treatment Isotype IgG Isotype IgG MF-1 Isotype IgG 35 Tumor 3 R1 cells + 25 2 15 1 ** * ** 5 CON -shcon -shcon -shcon + MF-1 -sh Supplementary Figure 7. Flow cytometric analysis showing the effects of MF-1 treatment and - knockdown on reducing the expression of R1 + cells in the tumor of mice bearing - overexpressing tumor xenografts (female 6-8 week old nude mice, n = 3 per group)..
CXCL5 in Lung Mice bearing xenograft of CON -sh Supplementary Figure 8. Immunohistochemical detection of CXCL5 in the lung tissue of mice bearing indicated tumor xenografts. Scale bar, 2 µm.
Fig. 1c Fig. 1e (ng/ml) 5 1 5 1 α-sma α-sma α-sma α-sma Fig. 2a Fig. 3c 1 2 3 4 5 6 7 8 9 1 11 N T N T N T N T N T N T N T N T N T N T N T - 25 kd mir-127-5p mir-29c.4.8.4.8 1 2 3 4 5 6 7 8 9 1 11 NEF CAF NEF CAF NEF CAF NEF CAF NEF CAF NEF CAF NEF CAF NEF CAF NEF CAF NEF CAF NEF CAF α-sma Supplementary Figure 9. Full blots of indicated figures.
Fig. 3d Fig. 3f mir-29c mir-29c Mimic (nm) 25 5 25 5 mir-29c Inhibitor (nm) 25 5 25 5 Fig. 4c p53 p53 Supplementary Figure 1. Full blots of indicated figures.
Fig. 4f Fig. 4g (ng/ml) P53 -/- MEF 5 1 p53 p53 p53 Supplementary Fig.1c KYSE15 sirna Id2 sirna Id3 sirna Id4 sirna sirna (Nm) 5 1 5 1 5 1 5 1-25 kd - 15 kd Id2-15 kd Id4 Id3-1 kd - 1 kd -1 kd - 15 kd - 25 kd - 25 kd KYSE27 sirna Id2 sirna Id3 sirna Id4 sirna sirna (Nm) 5 1 5 1 5 1 5 1-25 kd - 25 kd Id2 Id3 Id4-25 kd - 25 kd - 15 kd - 15 kd - 15 kd Supplementary Figure 11. Full blots of indicated figures.
Supplementary Fig. 1e Supplementary Fig. 1h CM from PIGF CON PIGF CON (ng/ml) p-akt 5 1-7 kd p-akt 5 1-7 kd - 15 kd AKT -7 kd AKT -7 kd FGF - 1 kd FGF -1 kd - 25 kd p-mdm2 p53-1 kd - 7 kd p-mdm2 p53-1 kd -7 kd Supplementary Figure 12. Full blots of indicated figures.
Supplementary Table 1. Correlation between / expression levels and clinicopathological parameters in patients with ESCC. Variable n Low / Low High / High P value a Age (years) 55 13 6 7 55 51 26 25 1 Gender Female 16 8 8 Male 48 24 24 1 Histological grade Poorly differentiated 14 8 6 Moderately/well differentiated 48 23 25.762 T-Stage 1/2 14 12 2 3/4 5 2 3.5 N-Stage N 32 18 14 N1 32 14 18.454 M-Stage M 58 32 26 M1 6 6.24 Pathologic stage Stages I & II 26 18 8 Stages III & IV 38 14 24.21 a Fisher s exact test. Statistical significance (P <.5) is shown in bold.
Supplementary Table 2. Cox proportional hazard regression analyses for overall survival. Clinical features Univariate analysis Multivariate analysis HR (95 % CI) P value HR (95 % CI) P value Age 1.13 (.545-1.88).968 - - Gender 1.211(.681-2.154).515 - - Differentiation 1.453(.811-2.62).29 - - pt stage 2.342(1.23-4.562).12 - - pn stage 1.748(1.44-2.926).34 - - pm stage 3.455(1.414-8.439).7 - - Pathological stage 2.476(1.438-4.263).1 2.269(1.37-3.942).4 / expression 1.459 (1.11-1.918).7 1.372 (1.39-1.812).26 HR Hazard ratio, CI Confidence interval Statistical significance (P <.5) is shown in bold.
Supplementary Table 3. Primers used in RT-PCR for 4 putative mirnas that bind with 3 UTR of. mirnas mir15a-5p mir29c mir13a mir125a-3p mir125a-5p mir127-5p mir134-5p mir14-5p mir185-5p mir186-5p mir199a-5p mir2b mir25-5p mir299 mir3 mir329 mir33-3p mir331-3p mir339-5p mir361-5p mir374a mir383-5p mir41 mir495 mir53-5p mir516a-3p mir593 mir874 mir939-5p mir365 mir3668 mir398 mir427 mir4279 mir4455 mir4481 mir4524a-5p mir4731-5p mir4742-3p mir4782-5p Primer sequence CTAGCAGCACATAATGGTTTGTGA GCTAGCACCATTTGAAATCGGTTA GCAGCATTGTACAGGGCTATGA GTGAGGTTCTTGGGAGCCA CCCTGAGACCCTTTAACCTGTGA CTGAAGCTCAGAGGGCTCTGAT GACTGGTTGACCAGAGGGGA GCCAGTGGTTTTACCCTATGGTAG GGAGAGAAAGGCAGTTCCTGAA CAAAGAATTCTCCTTTTGGGCT AGTGTTCAGACTACCTGTTCA AATACTGCCTGGTAATGATGAA CTTCATTCCACCGGAGTCTGA TATGTGGGATGGTAAACCGCTT GCCTATACAAGGGCAGACTCTCTCT GCAACACACCTGGTTAACCTCTTT GCACACGGCCTGCAGAGA CCCTGGGCCTATCCTAGAA GTCCTCCAGGAGCTCACG GCTTATCAGAATCTCCAGGGGTAC TTATAATACAACCTGATAAGTGAA GAGATCAGAAGGTGATTGTGGCT GCGAATATAACACAGATGGCCTGTA AACAAACATGGTGCACTTCTTA AGCGGGAACAGTTCTGCAG TGCTTCCTTTCAGAGGGTAAA GCTGTCTCTGCTGGGGTTTCT TGGCCCGAGGGACCGA GCTGAGGCTCTGGGGGTG CGCAGGTGTGTCTGTAGAGTCC GCGCAATGTAGAGATTGATCAAAAT GCGAGCAATGTAGGTAGACTGTTT AGGGAGTCAGGGGAGGGC GCTCTCCTCCCGGCTTC CGCAGGGTGTGTGTGTTTTT CGGAGTGGGCTGGTGGTTA GATAGCAGCATGAACCTGTCTCA GGGGGCCACATGAGTGTG TCTGTATTCTCCTTTGCCTGCAG GCCTTCTGGATATGAAGACAATCAA
Supplementary Table 4. Primer sequences used for testing p53 mutation. Exon Primer Sequence (5-3 ) exon1 Forward CACAGCTCTGGCTTGCAGA Reverse AGCGATTTTCCCGAGCTGA exon2 Forward AGCTGTCTCAGACACTGGCA Reverse GAGCAGAAAGTCAGTCCCATG exon3-4 Forward AGACCTATGGAAACTGTGAGTGGA Reverse GAAGCCTAAGGGTGAAGAGGA exon5-6 Forward CGCTAGTGGGTTGCAGGA Reverse CACTGACAACCACCCTTAAC exon7 Forward CTGCTTGCCACAGGTCTC Reverse TGGATGGGTAGTAGTATGGAAG exon8-9 Forward GTTGGGAGTAGATGGAGCCT Reverse GGCATTTTGAGTGTTAGACTG exon1 Forward CTCAGGTACTGTGTATATACTTAC Reverse ATACTACGTGGAGGCAAGAAT exon11 Forward TCCCGTTGTCCCAGCCTT Reverse TAACCCTTAACTGCAAGAACAT
Supplementary Table 5. Primers used for generating pgl3-mir29c promoter mutations. Primer BS1-F BS1-R BS2-F BS2-R BS3-F BS3-R Sequence 5'-tcactcccttaccttccacataagaacagctcggacagacagat-3' 5'-atctgtctgtccgagctgttcttatgtggaaggtaagggagtga-3' 5'-gccctgtactaaatgttgcatcttgatttcttgagccatatgggct-3' 5'-agcccatatggctcaagaaatcaagatgcaacatttagtacagggc-3' 5'-tggccttgatggcactaaagaatgacccccagaaaggtcc-3' 5'-ggacctttctgggggtcattctttagtgccatcaaggcca-3'
Supplementary Table 6. Primer sequences used in ChIP-qPCR assay. Primer ChIP-BS1-F ChIP-BS1-R ChIP-BS2-F ChIP-BS2-R ChIP-BS3-F ChIP-BS3-R Sequence 5'-tgtcttcctttcggcacttc -3' 5'-tccaagttgtttgggtgtca -3' 5'-gggctgccctgtactaaatg -3' 5'-gtggtcagggtgaggaacat -3' 5'-ggttgagcatgccaataaaga -3' 5'-ggcaaatgggatttaagtaaccaga -3'