ChIP assays were performed using antibodies against the indicated histone modifications and analyzed by quantitative PCR. Genomic DNA fragments covering the −3.5 kb to + 3.5 kb region with respect to the transcription start site of DACT1, 2 and 3 for PCR analysis were indicated with numbers. The relative enrichments (bound/input) (Mean ± SD.of triplicate measurement) encompassing the indicated regions is shown for each histone marks at each gene locus.
A: Histone marks at the DACT1-3 loci in HT-29 cells.
B: Enrichments of H3K27me3 and H3K4me3 at the DACT3 locus in SW480 and RKO cells.
C: Enrichments of H3K27me3 and H3K4me3 at the DACT3 locus in normal human intestinal epithelial cells (FHs 74 Int), non-cancerous breast epithelial MCF10A cells and lung fibroblast MRC5 cells.

A: Immunoblot analysis of DACT3, DVL2, total β-catenin, and non-phosphorylated β-catenin (Active-β-catenin) in SW480 and DLD1 cells treated with DZNep (5 µM), TSA (100 nM) or both.
B: Immunofluorescent images of DLD1 and SW480 cells treated with DZNep/TSA. β-catenin staining is red, nuclear staining is green (DRAQ5). Scale bars represent 10 µm.
C: Indicated colorectal cancer cell lines were treated as above; RNA was harvested and subjected to array analysis. Changes of expression of known Wnt/β-catenin target genes were shown using gene clustering program. Green represents down-regulated genes.
D: DLD1 and HT29 cells were treated as in A, and cell death was determined by PI staining and FACS analysis. The data represent Mean ± SD of three independent experiments.
E: HT29 cells were treated as in A, and Caspase 3 activity was measured by FACS analysis.
F: HT29 cells were treated as in A, followed by JC-1 staining and FACS analysis.

A: Exogenously expressed DACT3 associates with Dvl2. SW480 cells were transiently transfected with the indicated Flag or HA-tagged expression constructs. DACT3 was immunoprecipitated with anti-Flag and the immunoprecipitates were probed with anti-HA antibody. Reciprocal coimmunoprecipitation between DACT3 and Dvl2 is shown on the lower panel.
B: Western blot analysis of HA-tagged Dvl2 protein levels in SW480 cells co-transfected with Flag-DACT3 or an empty vector.
C: Immunofluorescent images of SW480 cells transfected with Myc-tagged DACT1 or DACT3 expression construct for 72h. Myc-tagged DACT1 or DACT3 and β-catenin levels were detected by staining with anti-myc (green) and anti-β-catenin (red). Nuclei were stained by DRAQ5 (blue). Arrows indicate transfected cells expressing DACT3-myc. Scale bar represents10 µm.
D: Colony formation assay showing ecotopic expression of DACT3 suppresses colon cell growth. DLD1 cells were transfected with a DACT3 expression vector or the empty vector, and selected for 12 days with Zeocin. The number of colonies relative to the control in triplicates is shown on the right (error bar: Mean ± SD).

A: Hierarchical clustering of Wnt inhibitors (upper) and Wnt/β-catenin target genes (lower) in human colorectal tumors (T) and matched normal mucosa (N).
B: RT-PCR analysis of SFRP1 and DACT1, 2 and 3 from random-selected 8 pairs of human colorectal tumor and matched mucosa.
C: Methylation status of SFRP1 and DACT3 in 8 tumor determined by methylation specific PCR (MSP) assay. M1, M2, M3 and M4 represent the examined PCR regions covering the entire CpG island of DACT3 promoter.
D: RT-PCR analysis of SFRP1 and DACT1, 2 and 3 in a panel of colorectal cancer cell lines compared to the normal tissue.
E: Methylation status of SFRP1 and DACT1, 2 and 3 in colorectal cancer cell lines.
F: Determination of methylation status of CpG sites by sequencing of bisulfite-modified DNA from normal and colorectal cancer cell lines. Arrows indicate the transcription start sites. Open cycles represent unmethylated CpGs; closed cycles denote methylated CpGs.
G: MSP analysis of DACT1, 2 and 3 promoters in HCT116 cells untreated or treated with 5-AzaC and in HCT116-DNMT1/DNMT3b−/− (DKO) cells.
H: RT-PCR analysis of SFRP1 and DACT1, 2 and 3 in HCT116 and DLD1 cells treated with 5-AzaC (5 uM) for 3 days and in DKO cells.

A: DACT3 mRNA expression analysis using Illumina Beadarray in DLD1 cells treated with 5 µM DZNep, 5 µM 5-AzaC, 200 nM TSA or their combinations. The data shown represents Mean ± SD of three independent experiments.
B: Indicated colorectal cancer cell lines were treated with DZNep, TSA or both. RNA was harvested and subjected to array analysis. Changes of expression of known Wnt/β-catenin antagonists are shown.
C: Immunoblotting analysis of bulk histone modifications induced by DZNep, TSA or both in HT29 cells and SW480 cells.
D: ChIP analysis of indicated the changes of histone marks at the DACT1 and DACT3 loci in HT29 cells untreated or treated with DZNep/TSA. The values represent the normalized enrichments against the changes of a low background region before and after the drug treatment. The data represent Mean ± SD of three independent experiments.

A: Two DLD1 stable clones expressing DACT3 shRNA (DACT3-sh1 and DACT3-sh2) or DLD1 cells expressing a non-targeting control shRNA (NC) were treated with DZNep, TSA or both. DACT3 and actin mRNA levels were determined by RT-PCR analysis; DVL2, active β-catenin and total β-actin protein levels were assessed by immunoblotting.
B: NC, DACT3-sh1 and DACT3-sh2 cells were treated as above and apoptosis was determined by PI staining and FACS analysis. The data represent Mean ± SD of three independent experiments. **, p < 0.01.
C: SW480 cells transfected with DACT3 SmartPool siRNA (DACT3-SP) and an independent DACT3 siRNA (DACT3-2) were treated as in A. The data represent Mean ± SD of three independent experiments. **, p < 0.05. Apoptosis, DACT3 mRNA levels, DVL2, active β-catenin protein levels, and apoptosis were determined as previously.
D: SW480 cells transfected with DACT1 siRNA were untreated or treated with DZNep/TSA, and harvested for mRNA and apoptosis analysis as above. The data represent Mean ± SD of three independent experiments.