Journal: International journal of oncology

Article Title: GATA-binding protein 1 is a novel transcription regulator of peroxiredoxin 5 in human breast cancer cells.

doi: 10.3892/ijo.2011.1236

Figure Lengend Snippet: Figure 1. Identification of the Prx5 promoter region in MCF7s cells. (A) MCF7s cells were transfected with a series of Prx5 promoter reporter constructs. The Renilla phRL-CMV vector was used to normalize the transfection efficiency. The reporter plasmid pGL3-p3000 indicated the size of the Prx5 promoter region inserted to the pGL3-basic vector. Luciferase activity of the pGL3-basic empty vector was used as a control. (B) The 500 bp region between pGL3-p3000 and pGL3-p2500 was subcloned into the pGL3-basic vector to confirm the recovery of luciferase activity. The reporter plasmid pGL3-F500 indicated the region between pGL3-p3000 and pGL3-p2500. (C) The 500 bp region upstream of the Prx5 translation start site subcloned as pGL3-F500 was analyzed using a promoter prediction program. The translation start codon, ATG, is indicated in blue. The sequences denoted in red span the 500-bp nucleotides in the pGL3-F500 reporter plasmid. (D) The three binding sites of the predicted transcription factors were targeted for site-directed mutagenesis. These mutational constructs were trans fected into MCF7s cells. The Renilla phRL-CMV vector was co-transfected to normalize the transfection efficiency.

Article Snippet: Small interfering RNA (siRNA) transfection. siRNAs targeting either GATA1 or Prx5 were obtained from Santa Cruz Biotechnology. siRNA was transfected into MCF7-Adr or MDA-MB-231 cells using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions a day after cells were seeded in 100 cm2 culture dishes.

Techniques: Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Control, Binding Assay, Mutagenesis

Journal: International journal of oncology

Article Title: GATA-binding protein 1 is a novel transcription regulator of peroxiredoxin 5 in human breast cancer cells.

doi: 10.3892/ijo.2011.1236

Figure Lengend Snippet: Figure 2. GATA1-mediated modulation of both gene expression and promoter activity of Prx5. (A) GATA1 siRNA (13 nM) was transfected into MDA-MB- 231 cells, which have a high level of endogenous GATA1 expression, and Prx5 expression in these cells was confirmed using real-time quantitative RT-PCR and compared to those treated with vehicle control (‘MDA-MB-231 con’ in the figure). GAPDH was used as a control for normalization. (B) MDA-MB-231 cells were seeded in a 6-well plate. Twenty-four hours after transfection with either pGL3-F500 alone, a combination of pGL3-F500 and GATA1 siRNA (30 nM), or pGL3-F500 containing a mutation in the GATA1 binding site (pGL3-F500-GATA1 mt), the cells were harvested and their luciferase activity was determined. (C) pGL3 vectors containing various portions of the Prx5 promoter region were transfected into MCF7s cells and luciferase activities were measured as described in (B). Figure 3. Direct binding of GATA1 to the Prx5 promoter in cell lysates and cultured breast cancer cells. (A) A ChIP assay was performed in MDA-MB- 231 cells, which have high endogenous GATA1 expression levels, and in MCF7s cells using the antibodies and primer pairs described in ‘Materials and methods’. Binding affinity was measured by PCR. Binding affinity of mouse normal IgG was used as a negative control. An arrow indicates the PCR product corresponding to GATA1 binding site in the Prx5 promoter region. (B) EMSA was carried out in MDA-MB-231 cells using radiolabeled oligo nucleotides containing the wild-type or mutated sequences corresponding to the GATA1 binding site of the Prx5 promoter. Unlabeled oligonucleotides containing either wild-type or mutant GATA1 binding sites were used as a specific competitor. (C) EMSA was carried out in MCF7s cells using radio labeled oligonucleotides containing the GATA1 binding site of the Prx5 promoter.

Article Snippet: Small interfering RNA (siRNA) transfection. siRNAs targeting either GATA1 or Prx5 were obtained from Santa Cruz Biotechnology. siRNA was transfected into MCF7-Adr or MDA-MB-231 cells using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions a day after cells were seeded in 100 cm2 culture dishes.

Techniques: Gene Expression, Activity Assay, Transfection, Expressing, Quantitative RT-PCR, Control, Mutagenesis, Binding Assay, Luciferase, Cell Culture, Negative Control, Labeling

Journal: International journal of oncology

Article Title: GATA-binding protein 1 is a novel transcription regulator of peroxiredoxin 5 in human breast cancer cells.

doi: 10.3892/ijo.2011.1236

Figure Lengend Snippet: Figure 4. Protection of cells from hydrogen peroxide-induced apoptosis via Prx5 expression (A) Prx5 siRNA was transfected into MCF7-Adr cells, which have high levels of endogenous Prx5. An inset picture shows the relative expression of Prx5 in cells treated with siRNA or no siRNA (control). FACS analysis was performed to measure H2O2-induced apoptosis in MCF7-Adr cells treated with Prx5 siRNA. Cells not exposed to H2O2 were used as a control to confirm annexinV staining. (B) Mean fluorescence intensities in MCF7-Adr cells with high or low Prx5 expression produced similar FACS histogram patterns. (C) GATA1 siRNA was transfected to MDA-MB-231 cells and the expression of GATA1 was detected by RT-PCR compared to samples treated with no siRNA (control). FACS analysis was performed as described in (A). (D) Mean fluorescence intensity in MDA-MB-231 cells with high or low GATA1 expression produced similar FACS histogram patterns. All experiments were performed in duplicate.

Article Snippet: Small interfering RNA (siRNA) transfection. siRNAs targeting either GATA1 or Prx5 were obtained from Santa Cruz Biotechnology. siRNA was transfected into MCF7-Adr or MDA-MB-231 cells using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions a day after cells were seeded in 100 cm2 culture dishes.

Techniques: Expressing, Transfection, Control, Staining, Fluorescence, Produced, Reverse Transcription Polymerase Chain Reaction

Journal: International journal of oncology

Article Title: GATA-binding protein 1 is a novel transcription regulator of peroxiredoxin 5 in human breast cancer cells.

doi: 10.3892/ijo.2011.1236

Figure Lengend Snippet: Figure 5. Modulation of apoptotic/anti-apoptotic proteins in response to knockdown of either Prx5 or GATA1 expression. Samples designated as ‘Non’ represent those treated with no siRNA. Control samples that were not chal lenged with H2O2 are expressed as ‘No treat’. (A) Prx5 siRNA was transfected into MCF7-Adr cells. The cells were treated with H2O2 to induce apoptosis 24 h after transfection. The expression of Bcl-2, an anti-apoptotic protein, and Bax, a pro-apoptotic protein, were detected by Western blotting. β-Actin was used as a loading control. (B) GATA1 siRNA was transfected into MDA-MB-231 cells. Protein levels were determined as described in (A).

Article Snippet: Small interfering RNA (siRNA) transfection. siRNAs targeting either GATA1 or Prx5 were obtained from Santa Cruz Biotechnology. siRNA was transfected into MCF7-Adr or MDA-MB-231 cells using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions a day after cells were seeded in 100 cm2 culture dishes.

Techniques: Knockdown, Expressing, Control, Transfection, Western Blot