Representative results of <i>DACH1</i> suppresses esophageal cancer growth <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Growth curves represent CCK-8 assay results for <i>DACH1</i> expressed cells and unexpressed cells.Points, mean of four independent experiments; bars, SEM. **, <i>P</i><0.01, Student’s <i>t</i> test. (B) Representative results of colony formation in <i>DACH1</i> expressed and unexpressed KYSE510 and KYSE150 cell lines. Columns, mean of four independent experiments; bars, SEM. *, <i>P</i><0.05 versus controls by using the Student’s <i>t</i> test. (C) Representatives results of xenograft tumors in nude mice for <i>DACH1</i> expressed and unexpressed KYSE510 cells. (D) Growth curves represent tumor size in <i>DACH1</i> expressed and unexpressed KYSE510 cells xenograft mice in different time. Points, mean of 5 mice; bars, SEM.*, <i>P</i><0.01, Student’s <i>t</i> test. (E) Representative results of tumor weight in <i>DACH1</i> expressed and unexpressed KYSE510 cells xenograft mice in different time. Columns, mean of 5 mice; bars, SEM. *, <i>P</i><0.01, Student’s <i>t</i> test. (F) Representive DACH1 expression results detected by IHC for <i>DACH1</i> expressed and unexpressed KYSE510 cells xenograft. DACH1 expression was found in <i>DACH1</i> expressed KYSE510 cell xenograft. (right). Magnification: upper phase, X200; lower phase, X400.</p

Representative results of <i>DACH1</i> expression and methylation in esophageal cancer cells.

<p>(A) <i>DACH1</i> expression level detected by RT-PCR in esophageal cancer cell lines. (B) Methylation status in promoter region; IVD: <i>in vitro</i> methylated DNA, used as methylation control; NL: normal blood lymphocyte DNA, used as unmethylation control; U: unmethylated alleles; M: methylated alleles. (C) BSSQ of <i>DACH1</i> promoter region (−426 bp to −140 bp) in KYSE150, KYSE510, TE8 and KYSE140 cells; double-headed arrow: MSP PCR product, spanning 130 bp. Filled circles: methylated CpG sites; open circles: unmethylated CpG sites.</p

Clinicopathologic characteristics and DACH1 methylation status of 104 patients with esophageal squamous cell carcimoma.

<p>*<i>P</i> values are obtained from chi-square test.</p>#<p>Statistical significance is indicated by <i>P</i><0.05.</p

Representative results of <i>DACH1</i> methylation and expression in primary esophageal cancer.

<p>(A) Representative MSP results of <i>DACH1</i> methylation status in normal esophageal mucosa (NE), esophageal dysplasia (ED) and esophageal cancer (EC). (B) <i>DACH1</i> methylation frequency in NE, ED1, ED2 and ED3, and EC. The frequency of methylated <i>DACH1</i> were plotted according to histological grade and analyzed using chi-square test. **, <i>P</i><0.01. (C) Representative IHC results for DACH1 expression in primary esophageal cancer (left) and adjacent tissues (right); upper phase, X200; lower phase, X400. (D) DACH1 expression level in 30 cases matched primary cancer and adjacent tissue samples; box plot: represents DACH1 expression level; horizontal line: represent the median level; the top and bottom line of the boxes represent 75% and 25% expression level, respectively; vertical bars represent different expression level. **, <i>P</i><0.01 versus adjacent tissue samples by using Wilcoxon signed-rank test. (E) The association of <i>DACH1</i> methylation and loss/reduced expression in 30 cases ESCC. **, <i>P</i><0.01, Spearman’s rank correlation coefficient.</p

Flow cytometry and western blot results.

<p>(A) Flow cytometry results show: the cell phase distribution in DACH1 unexpressed and expressed KYSE510 and KYSE150 cells. Columns, mean of three independent experiments; bars, SEM. *, <i>P</i><0.05; **, <i>P</i><0.01, Student’s <i>t</i> test. (B) Western blot results show: expression of G1/S check point related genes in DACH1 unexpressed and expressed KYSE510 and KYSE150 cells, β-Actin was used as a loading control.</p

Effect of <i>DACH1</i> on TGF-β signaling in human esophageal cancer cells.

<p>(A) Smad-binding elements (SBE)-4 Luc reporter activities in KYSE510 and KYSE150 cells. Columns, mean of three independent experiments; bars, SEM. (B) The expression level of TGF-β signaling downstream genes in <i>DACH1</i> expressed cells and unexpressed cells, β-Actin was used as a loading control. (C) The efficiency of siRNAs targeting on <i>DACH1</i> in KYSE140 cells. (D) The expression level of TGF-β signaling downstream genes in <i>DACH1</i>-siRNA KYSE140 cells and control group, β-Actin was used as a loading control.</p