cdx2 sirna duplex  (Santa Cruz Biotechnology)

Journal: Archives of toxicology

Article Title: Aryl hydrocarbon Receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells

doi: 10.1007/s00204-016-1880-y

Figure Lengend Snippet: Expression of A) ATF3, B) IDO1, and C) CDX2 in AhR- and TLR-activated MoDCs. Cells were treated with 10 nM TCDD, 100 nM FICZ, 50 μM I3C, or 50 μM kyn in absence or presence of LPS (50 ng/ml), CL075 (5 μg/ml), or poly(I:C) (5 μg/ml) for 24h. Results from real-time PCR are presented as mean ± SEM and the y-axis represents mRNA expression level as fold increase above control. a significantly higher than non-activated DC control, p < 0.05; b significantly higher than TLR- and AhR-activated MoDCs, p < 0.05

Article Snippet: Cdx2 siRNA duplex (sc-43680) and Control siRNA duplex (sc-37007) were from Santa Cruz Biotechnology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Archives of toxicology

Article Title: Aryl hydrocarbon Receptor signaling modifies Toll-like receptor-regulated responses in human dendritic cells

doi: 10.1007/s00204-016-1880-y

Figure Lengend Snippet: Effect of gene silencing of AhR, RelB, and CDX2 on the expression of IL-1β and IL-12A. Schematic illustration of the promoter of the human A) IL-1β gene. Positions of the two DRE consensus elements, one putative NF-κB recognition site, and one RelBAhRE binding site are presented. B) The promoter of the human IL-12A gene containing two DRE consensus elements and two CDX2 binding sites. C) Western blot analysis of AhR, RelB, and CDX2 protein levels 24h post-transfection of cells with the indicated siRNAs. D) IL-1β and E) IL-12A mRNA expression analyses after treatment with TCDD and LPS or co-treatment with LPS plus TCDD for 24 h as analyzed by real-time PCR. Total RNA was prepared 48 h post-transfection with either a scrambled siRNA or a specific siRNA targeted against AhR, RelB, or CDX2. After 24 h transfected cells were treated with 10 nM TCDD or 50 ng/ml LPS for 24 h. Results are presented as mean ± SEM and the y-axis represents mRNA expression level as fold increase above control. a significantly higher compared to control, p < 0.05; b significantly higher than cells treated with TCDD or LPS alone, p < 0.05; c significantly lower than cells treated with LPS alone, p < 0.05; d significantly different compared to cells transfected with scrambled siRNA, p < 0.05

Article Snippet: Cdx2 siRNA duplex (sc-43680) and Control siRNA duplex (sc-37007) were from Santa Cruz Biotechnology.

Techniques: Expressing, Binding Assay, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Reg IV Is a Direct Target of Intestinal Transcriptional Factor CDX2 in Gastric Cancer

doi: 10.1371/journal.pone.0047545

Figure Lengend Snippet: A: Western blot analysis of CDX2, Reg IV, and β-actin and qRT-PCR analysis of REG4 in 9 GC cell lines. B: Western blot analysis of CDX2, Reg IV, and β-actin and qRT-PCR analysis of REG4 in HT-29/PGS-CDX2, HT-29/PGS-neo, SW480/PGS-CDX2, and SW480/PGS-neo. C: Western blot analysis of Reg IV and β-actin and qRT-PCR analysis of REG4 in HT-29/CDX2-ER. Time course of REG4 gene induction in response to activation of a CDX2-ER fusion protein by 4-OHT was analyzed. D: Western blot analysis of CDX2, Reg IV, and β-actin and qRT-PCR analysis of REG4 in HSC-39 cells transfected with CDX2 siRNA (siRNA1 and siRNA2) and the negative control siRNA. The units of REG4 mRNA expression level are arbitrary. P values were calculated using Student’s t-test. * N.S. = not significant.

Article Snippet: Two siRNA duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′, CDX2 siRNA1; and 5′-AAGCCUCAGUGUCUGGCUCUG-3′, CDX2 siRNA2) and a nonsilencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized (Qiagen).

Techniques: Western Blot, Quantitative RT-PCR, Activation Assay, Transfection, Negative Control, Expressing

Journal: PLoS ONE

Article Title: Reg IV Is a Direct Target of Intestinal Transcriptional Factor CDX2 in Gastric Cancer

doi: 10.1371/journal.pone.0047545

Figure Lengend Snippet: Localization of regulatory elements and CDX2 binding sites in the 5′-flanking region of the REG4 gene. A: Schematic representation of the 5′-flanking region of the REG4 gene. The location and sequence of 4 consensus CDX2-binding sites in the 5′-flanking region of REG4 (i.e., sites A, B, C, and D) is indicated. B: Schematic representation of REG4 reporter gene constructs. The REG4 genomic DNA sequences present in the reporter gene vectors are indicated. Key sequences for REG4 transcription reside between base pairs −634 and −116. Reporter assays with the series of REG4 deletion constructs were performed in the CDX2-expressing GC cell line, HSC-39. The luciferase activity of the empty pGL4.10 basic vector was assigned a value of 1. The reporter assays were performed in triplicate, and mean and SD values of luciferase activity are shown. C: Localized mutations in the candidate CDX2-binding sites (i.e., sites A, B, C, and D) were introduced into the −2019/+58 construct, and the series of constructs generated is shown. The CDX2 candidate binding site designated as “C” plays critical roles in REG4 transcription. Reporter assays were performed in CDX2-expressing GC cell line, HSC-39. The activity of the pGL4.10 basic vector was assigned a value of 1. Assays were performed in triplicate. Mean and SD luciferase activity values are shown.

Article Snippet: Two siRNA duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′, CDX2 siRNA1; and 5′-AAGCCUCAGUGUCUGGCUCUG-3′, CDX2 siRNA2) and a nonsilencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized (Qiagen).

Techniques: Binding Assay, Sequencing, Construct, Expressing, Luciferase, Activity Assay, Plasmid Preparation, Generated

Journal: PLoS ONE

Article Title: Reg IV Is a Direct Target of Intestinal Transcriptional Factor CDX2 in Gastric Cancer

doi: 10.1371/journal.pone.0047545

Figure Lengend Snippet: A: Schematic representation of the 5′-flanking region of the REG4 gene. The location of 4 consensus CDX2-binding sites in the 5′-flanking region of REG4 (sites A, B, C, and D) and PCR primers ( REG4 Primer 1, 2, 3, 4, 5, and 6) are indicated. B: CDX2 binding to REG4 promoter region shown by ChIP. Bulk (input) DNA was prepared as well as DNA isolated from ChIP with anti-CDX2 monoclonal antibody or mouse IgG. qPCRs were performed in triplicate for each sample primer set, and the mean and SD of the three experiments was calculated. C: ChIP analysis of H3K27me3 enrichment in the REG4 gene promoter. ChIP enrichment was measured using qPCR. qPCRs were performed in triplicate for each sample primer set, and the mean and SD of the three experiments was calculated. P values were calculated using Student′s t-test. * N.S. = not significant.

Article Snippet: Two siRNA duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′, CDX2 siRNA1; and 5′-AAGCCUCAGUGUCUGGCUCUG-3′, CDX2 siRNA2) and a nonsilencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized (Qiagen).

Techniques: Binding Assay, Isolation

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: CDX2 activates MDR1 expression in HT-29 cells. (A, upper) A monoclonal anti-CDX2 antibody detects the roughly 40-kD CDX2 protein in HT-29/PGS-CDX2 cells but not in HT-29/PGS-neo cells. (A, lower) Relative level of MDR1 gene expression in HT-29/PGS-CDX2 and HT-29/PGS-neo in Affymetrix microarray studies. (B) Northern and Western blot analysis detects MDR1 transcripts and products in HT-29/PGS-CDX2 with low or absent MDR1 expression in HT-29/PGS-neo cells. In Western blot analysis, a mouse monoclonal anti-MDR1 antibody detects the roughly 170-kD MDR1 product in HT-29/PGS-CDX2 cells but not in HT-29/PGS-neo cells. (C) Expression of CDX2 and MDR1 in 13 CRC cell lines. In the indicated 13 colorectal cancer cell lines, Western blot analyses of MDR1 and CDX2 expression were performed using a mouse monoclonal antibody against human MDR1 and a mouse monoclonal antibody against human CDX2. The membranes were stripped and reprobed with a monoclonal antibody against β-actin to verify loading and transfer. Northern blot analysis of MDR1 expression was performed using an MDR1 cDNA probe. The membrane was stripped and reprobed with a GAPDH cDNA probe to verify loading and transfer.

Article Snippet: RNA interference Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Expressing, Gene Expression, Microarray, Northern Blot, Western Blot, Membrane

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: The MDR1 gene is a primary target of CDX2 action. (A) Time course of MDR1 gene induction in response to activation of a CDX2-ER fusion protein by 4-OHT. (B) Induction of MDR1 transcript in response to activation of a CDX2-ER fusion protein by 4-OHT is not inhibited by the protein synthesis inhibitor cycloheximide, but protein synthesis is blocked. (C) Inhibition of CDX2 expression by siRNA targeting leads to decreased MDR1 expression in CRC cell line DLD1. Assays were performed in triplicate, and mean and SD values are shown.

Article Snippet: RNA interference Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Activation Assay, Inhibition, Expressing

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: Localization of regulatory elements and CDX2 binding sites in the 5′-flanking region of the MDR1 gene. (A) Schematic representation of the 5′-flanking region of the MDR1 gene and MDR1 reporter gene constructs constructed. The location and sequence of four consensus CDX2-binding sites in the 5′-flanking region of MDR1 is indicated. The direction of the arrows indicates the strand on which the candidate CDX2-binding element was found (i.e., sense or antisense). The MDR1 genomic DNA sequences present in the reporter gene vectors are indicated. Localized mutations in the candidate CDX2-binding sites (i.e., site A and B) were introduced into the -4003/+50 construct as noted at the bottom of the panel, and the series of constructs generated is shown. (B) Key sequences for MDR1 transcription in CDX2-expressing cell lines reside between base pairs -4003 and -3414. Mean and SD values of the luciferase activity ratio in HT29/PGS-CDX2 cells to that in HT29/PGS-neo cells are shown. (C) CDX2 candidate binding sites “A” and “B” play critical roles in MDR1 transcription. All assays were performed in triplicate, and mean and SD values of luciferase activity ratio are shown.

Article Snippet: RNA interference Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Binding Assay, Construct, Sequencing, Generated, Expressing, Luciferase, Activity Assay

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: CDX2 binding to MDR1 promoter region shown by ChIP. (A) CDX2 function was activated in HT-29/CDX2-ER cells by treatment of the cells with 4-OHT, and the cells were harvested at the indicated time points. (B) Specificity of recovery of DNA fragments of MDR1 promoter region following ChIP with anti-CDX2 antibody was confirmed in Caco2, which has endogenous strong CDX2 expression. Assays were performed in triplicate, and mean and SD values are shown.

Article Snippet: RNA interference Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Binding Assay, Expressing

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: CDX2 and MDR1 expressions are well correlated in human colon epithelium and stomach cancer tissues. Immunohistochemistry was performed on formalin-fixed and paraffin-embedded tissues with anti-CDX2 monoclonal antibody (A, B, left) and with anti-MDR1 monoclonal antibody, C494 (A, B, right) in (A) human colon epithelium and (B) stomach cancer tissue.

Article Snippet: RNA interference Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Immunohistochemistry

Journal: Cancer research

Article Title: CDX2 regulates Multidrug Resistance 1 gene expression in malignant intestinal epithelium

doi: 10.1158/0008-5472.CAN-09-4701

Figure Lengend Snippet: HT29 cells ectopically expressing CDX2 have MDR1-dependent drug resistance. (A) Effect of chemotherapeutic drugs on HT29/PGS-CDX2 (open circle) and HT29/PGS-neo (closed circle) cell lines. (B) Effect of additional verapamil on vincristine and paclitaxel in HT29/PGS-CDX2 (open circle) and HT29/PGS-neo (closed circle) cell lines. (C) [IC50 (72h)] determined by MTT assay on HT29/PGS-CDX2 and HT29/PGS-neo cells. Co-treatment with verapamil significantly recovered the sensitivity of vincristine and paclitaxel on HT-29/PGS-CDX2 cells. The cytotoxic assays were performed in triplicate, and mean and SD values are shown.

Article Snippet: RNA interference Two small interfering RNA (siRNA) duplexes targeting CDX2 (5′-AACCAGGACGAAAGACAAAUA-3′; CDX2 siRNA-1) and 5′-AAGCCUCAGUGUCUGGCUCUG-3′; CDX2 siRNA-2) and a non-silencing siRNA duplex (5′-AAUUCUCCGAACGUGUCACGU-3′) were synthesized by Qiagen-Xeragon (Germantown, MD).

Techniques: Expressing, MTT Assay