Fig 1: The effect of DACH1 on breast cancer cell migration and invasion.a The protein abundance of DACH1 was examined by western blot in T47D, ZR-75-30, MCF-7 cell lines. b, c Scratch wound-healing assay assessing the effects of DACH1 overexpression or knockdown on the motility of ZR-75-30 and MCF-7 cells. d Transwell migration and invasion assays evaluating the effects of DACH1 on the motility and invasion of ZR-75-30 cells. Cell migration and invasion assays were performed in 24-well chambers without or with Matrigel. Cells were transfected with Flag-DACH1 or control vector and then inoculated into the upper chamber. After cultured 24 h, cells on the lower surface of the filter were stained and photographed. The scale bars stand for 100 µm. Experiments were performed in triplicates and the data are representative of three independent experiments.
Fig 2: DACH1 down-regulates the level of MMP9.a ZR-75-30 cells were transfected with Flag-DACH1 or control vector, and then subjected to the analysis of MMP9 and MMP2 expression by RT-PCR. b Western blot assay evaluating the protein level of MMP9 in ZR-75-30 cells transfected with Flag-DACH1 or control vector (the first three lines). The medium from this assay was subjected to gelatin zymography (the last line). c Breast cancer cells were transfected with MMP-Luc together with or without Flag-DACH1 (left) or sh-DACH1 (right). Luciferase activity was measured 24 h after transfection. d HEK293T cells were transfected with MMP-Luc along with 0, 200, 400, 600 ng Flag-DACH1, and the luciferase activity was measured 24 h after transfection. There were three independent experiments with three parallel wells each. The data are representative of three independent experiments. Data are presented as means ± SD (P < 0.05, significant; ns, not significant).
Fig 3: DACH1 interacts with p65 and c-Jun, respectively.a HEK 293 T cells transfected with Flag-DACH1 alone or both Flag-DACH1 and GFP-c-Jun were subjected to immunoprecipitation with anti-GFP antibody followed by Western blot with anti-Flag antibody. b HEK 293 T cells were transfected with Flag-DACH1 and GFP-c-Jun. After 24 h of transfection, DNA-protein complex were immunoprecipitated with anti-IgG or anti-GFP antibody, followed by re-immunoprecipitation with anti-Flag antibody. AP-1 binding site (region 3) was amplified by PCR. c HEK 293 T cells were transfected with pNF-?B-Luc and either DACH1 or p65, DACH1 and p65. Luciferase activity was measured 24 h after transfection. d HEK 293 T cells transfected with Flag-DACH1 and GFP-p65 were subjected to immunoprecipitation with anti-GFP antibody followed by Western blot with anti-Flag antibody or vice versa. e ZR-75-30 cells were subjected to immunoprecipitation with anti-p65 or anti-IgG antibody followed by Western blot with anti-DACH1 antibody. f HEK 293 T cells were transfected with Flag-DACH1 and GFP-p65. After 24 h of transfection, DNA-protein complex were immunoprecipitated with anti-IgG or anti-GFP antibody, followed by re-immunoprecipitation with anti-Flag antibody. NF-?B binding site (region 1) was amplified by PCR. There were three independent experiments with three parallel wells each. The data are representative of three independent experiments. Data are presented as means ± SD (P < 0.05, significant; ns, not significant). g, h ZR-75-30 cells were transfected with or without Flag-DACH1, and then subjected to the analysis of CCD1 or VEGFC expression by RT-PCR. i, j Up, the schematic structure of NF-?B binding site in VEGFC promoter or AP1 binding site in CCD1 promoter. Down, ZR-75-30 cells were transfected with Flag-DACH1. Cross-linked chromatin was extracted from the cells and subjected to immunoprecipitation with anti-Flag or IgG antibody. The DNA regions were amplified by PCR. IgG was used as a negative control.
Fig 4: Determination of DACH1 binding sites in MMP9 promoter.a HEK 293 T cells were transfected with a series of 5’-deletion constructs of human MMP9 promoter-reporter plasmids together with or without Flag-DACH1. After 24 h, the luciferase activities were measured. The bar graph shows the fold changes in relative luciferase activity normalized against the ß-galactosidase activity. There were three independent experiments with three parallel wells each. b ChIP assay showing the binding region of DACH1 in MMP9 promoter. Up, PCR amplified region of MMP9 promoter. Down, HEK 293 T cells were transfected with Flag-DACH1. Cross-linked chromatin was extracted from the cells and subjected to immunoprecipitation with anti-Flag antibody. The DNA regions were amplified by PCR. IgG was used as a negative control. c HEK 293 T cells were transfected with WT-MMP9-luc, NF-?B mut-MMP9-luc, AP-1 mut-MMP9-luc or dual-mut-MMP9-luc together with or without Flag-DACH1. Luciferase activity was measured 24 h after transfection. HEK 293 T cells were transfected with pNF-?B-Luc or pAP-1-luc along with or without Flag-DACH1. Luciferase activity was measured 24 h after transfection. Reporter-gene assay evaluating the regulation of site-specific mutational MMP9 luciferase reporter activity by DACH1 in HEK 293 T cells. The mutation of NF-?B: 5’-AATTCCCC-3’ to 5’-AATTGGCC-3’; the mutation of AP-1: 5’-CTGAGTCA-3’ to 5’-CTGTCACA-3’. d The luciferase activities of pNF-?B-Luc and pAP-1-luc were determined after transfected with Flag-DACH1 or control vector in HEK 293 T cells. There were three independent experiments with three parallel wells each. The data are representative of three independent experiments. Data are presented as means ± SD (P < 0.05, significant; ns, not significant).
Fig 5: DACH1 recruits HDAC1 to the NF-kB binding site.a HEK 293 T cells transfected with Flag-DACH1 alone or Flag-DACH1 and Myc-HDAC1 were subjected to immunoprecipitation with anti-Myc antibody followed by western blot with anti-Flag or anti-Myc antibody. b HEK 293 T cells transfected with GFP-p65 alone or together with Myc-HDAC1 were subjected to immunoprecipitation with anti-GFP antibody followed by Western blot with anti-Myc or anti-GFP antibody. c HEK 293 T cells transfected with GFP-p65 and Myc-HDAC1 along with or without Flag-DACH1 were immunoprecipitated with anti-GFP antibody followed by Western blot with anti-Myc, anti-GFP, anti-Flag antibody. d HEK 293 T cells transfected with the indicated plasmids were subjected to immunoprecipitation with anti-GFP antibody followed by Western blot with anti-acetylated lysine antibody. All cells were treated with TNFa (40 ng/ml) for 4 h before harvest. TNFa was used to activate the NF-?B signaling pathway and promote the acetylation level of p65. e HEK 293 T cells were transfected with MMP9-luc and either HDAC1 or DACH1, HDAC1 and DACH1. f HEK 293 T cells were transfected with the related plasmids for 24 h, and then the cell lysis were subjected to immunoprecipitation with anti-GFP antibody followed by western blot with anti-acetylated lysine antibody. g HEK 293 T cells were transfected with GFP-p65 along with or without Flag-DACH1. Cells were treated with or without TNFa (40 ng/ml) for 4 h before harvest. The lysis were subjected to immunoprecipitated with anti-GFP antibody followed by anti-acetylated lysine antibody. h HEK 293 T cells transfected with the related plasmids for 24 h and then the cells treated by TSA (1 µM) for 6 h before harvest were immunoprecipitated with anti-GFP antibody followed by western blot with anti-acetylated lysine antibody. i MCF7 cell invasion assays was performed in 24-well chambers with Matrigel. Cells were transfected with sh Ctrl, sh DACH1, sh MMP9 or sh DACH1 and sh MMMP9, and then inoculated into the upper chamber. After cultured 24 h, cells on the lower surface of the filter were stained and photographed. The scale bars stand for 100 µm. Luciferase activity was measured 24 h after transfection. Experiments were performed in triplicates and the data are representative of three independent experiments. The data are representative of three independent experiments. Data are presented as means ± SD (P < 0.05, significant).
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