Journal: PLoS ONE

Article Title: HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0164555

Figure Lengend Snippet: DNA oligo sequences used in this study.

Article Snippet: Four different constructs containing the HOXB4 promoter/enhancer region (4997-bp fragment) and in which each of the CDX2 binding sites, sequence a/cATAAAa/t[ ], were replaced with the EcoRI cleavage sequence, GAATTCG, were synthesized by Eurofins (Eurofins MWG Operon, Ebersberg, Germany).

Techniques: Sequencing, shRNA

Journal: PLoS ONE

Article Title: HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0164555

Figure Lengend Snippet: Fig 1A) Genome browser CDX2 ChIP-seq peaks from a dataset generated by Boyd et al.. Fig 1B) ChIP-PCR with primers covering the CDX2 binding region within the HOXB4 enhancer. Equal amounts of input DNA were used for CDX2 ChIP-PCR. Hemagglutinin (HA) was used as a negative control. The mean (n = 4) and SD are shown. * P < 0.05.

Article Snippet: Four different constructs containing the HOXB4 promoter/enhancer region (4997-bp fragment) and in which each of the CDX2 binding sites, sequence a/cATAAAa/t[ ], were replaced with the EcoRI cleavage sequence, GAATTCG, were synthesized by Eurofins (Eurofins MWG Operon, Ebersberg, Germany).

Techniques: ChIP-sequencing, Generated, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0164555

Figure Lengend Snippet: Fig 2A) Western blot analysis of CDX2 expression levels in SW480 cells treated with scrambled shRNA and cells in which CDX2 was knocked down. GADPH was used as a control to analyze equal amounts of total cellular protein. Fig 2B) The effects of CDX2 knock down on the promoter activities of reporter constructs containing the HOXB4 promoter alone or the HOXB4 promoter including the enhancer region. Fig 2C) Transient transfection of the HOXB4 promoter and enhancer reporter construct in SW480 cells with and without co-transfection of a CDX2 expression plasmid. The mean (n = 4) and SD are shown. * P <0.05 and **P<0.01

Article Snippet: Four different constructs containing the HOXB4 promoter/enhancer region (4997-bp fragment) and in which each of the CDX2 binding sites, sequence a/cATAAAa/t[ ], were replaced with the EcoRI cleavage sequence, GAATTCG, were synthesized by Eurofins (Eurofins MWG Operon, Ebersberg, Germany).

Techniques: Western Blot, Expressing, shRNA, Control, Knockdown, Construct, Transfection, Cotransfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: HOXB4 Gene Expression Is Regulated by CDX2 in Intestinal Epithelial Cells

doi: 10.1371/journal.pone.0164555

Figure Lengend Snippet: Fig 3A) Positions of the four potential CDX2 binding sites and the EMSA probes covering these sites. Fig 3B) Reporter gene assays with four different luciferase constructs containing the HOXB4 enhancer with wild-type CDX2 binding sites or four distinct reporter constructs containing individual mutations in the four CDX2 binding sites ( HOXB4 site-1, -2, -3 or 4). The mean (n = 4) and SD are shown. * P <0.05 and ** P <0.01. Fig 3C–3F) EMSAs with probes for the four different CDX2 binding sites (HOXB4 site-1, -2, -3 and 4). One hundred-fold unlabeled probe was added to the competition reactions as well as sucrase isomaltase (SI) or unspecific probe (unspec). Influenza hemagglutinin (HA) antibody (negative control) or CDX2 antibody (CDX2) was used in the supershift assay. Non-specific bands and supershifts indicated by black arrows.

Article Snippet: Four different constructs containing the HOXB4 promoter/enhancer region (4997-bp fragment) and in which each of the CDX2 binding sites, sequence a/cATAAAa/t[ ], were replaced with the EcoRI cleavage sequence, GAATTCG, were synthesized by Eurofins (Eurofins MWG Operon, Ebersberg, Germany).

Techniques: Binding Assay, Luciferase, Construct, Negative Control