Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH represses VSP genes. (A) Western blot of whole-cell extracts from K562 cells transiently transfected with increasing amounts of a plasmid expressing Myc-tagged PRH. Proteins were stained with a mouse anti-PRH antibody (top), the Myc9E10 monoclonal antibody (middle), and a lamin A/C antibody (bottom). (B) Vegfr-1 and Vegf mRNA levels in K562 cells 48 h after transfection with 10 μg pMUG1 or pMUG1-Myc-PRH. mRNA levels were determined by qRT-PCR using specific primers and compared to those of GAPDH. Values are means and standard deviations (SD) (n = 5). (C) Western blot of whole-cell extracts from K562 cells cotransfected with shRNA plasmids SVC shRNA, PRH shRNA 1, PRH shRNA 2, and PRH shRNA (1+2) (4). Proteins were stained with rabbit PRH antibody and lamin A/C antibody. (D) Prh, Vegf, Vegfr-1, and Vegfr-2 mRNA levels after shRNA (sh) cotransfection. Black bars represent SVC shRNA-targeted cells and gray bars PRH shRNA (1+2)-targeted cells. Values are means and SD (n = 5). (E) Western blot of whole-cell extract from K562 cells transfected with empty pMUG1 or pMUG1-MycPRH. Proteins were stained with VEGF antibody or lamin A/C antibody.

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Staining, Quantitative RT-PCR, shRNA, Cotransfection

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH represses VSP genes in conjunction with TLE proteins. (A) Vegf mRNA levels in K562 cells 48 h after transfection with 10 μg pMUG1 (−) or pMUG1 vectors expressing PRH (wild type [WT], PRH F32E, PRH L23A,L24A, or PRH N187A). mRNA levels were determined as for Fig. ​Fig.1.1. Values are means and SD (n = 3). (B) As for panel A except that relative levels of expression of Vegfr-1 are shown. Values are means and SD (n = 3). (C) Western blot of Myc-PRH expression in K562 cells 48 h after transfection with the vectors described for panel A. Whole-cell extracts were run on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) and stained with Myc and lamin A/C antibodies. (D) Vegf mRNA levels in K562 shRNA (sh) control cells and PRH shRNA cells 48 h after transfection with pMUG1-MycPRH (5 μg), pCMV2-TLE1(2.5 μg), or both plasmids were determined as for Fig. ​Fig.1.1. Values are means and SD (n = 3). (E) As for panel D except that relative levels of expression of Vegfr-1 are shown. Values are means and SD (n = 3).

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Transfection, Expressing, Western Blot, Staining, shRNA

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH binds to the Vegf, Vegfr-1, and Vegfr-2 promoters in cells. (A) Schematic of the human Vegfr-1 promoter (top line), with the positions of potential PRH binding sites indicated as bars. C1 to C5 indicate clusters of potential sites. The arrow corresponds to the first exon. Each inset panel shows the results of PCRs using the following templates: (1) input DNA, (2) DNA precipitated from cells expressing Myc-PRH with beads plus Myc antibody, (3) DNA precipitated from cells expressing Myc-PRH with beads plus normal IgG. The amplicons are indicated by solid bars. In the same experiment, primers within the actin promoter did not produce a product (left inset). (B) As for panel A except that the schematic and ChIP results are for the Vegfr-2 promoter. (C) As for panel A except that the schematic and ChIP results are for the Vegf promoter.

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Binding Assay, Expressing

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH directly represses the Vegf, Vegfr-1, and Vegfr-2 promoters. (A, left) Schematic representations of the Vegfr-1, Vegfr-2, and Vegf promoters indicating the position of the first exon for each gene (white arrows) and the promoter derivatives (black arrows) used in this study. (Middle) Relative promoter activity found in K562 cell extracts following transfection with Vegfr-1, Vegfr-2, or Vegf reporters. Five micrograms of each reporter and 5 μg of the β-galactosidase plasmid (pSV-lacZ) were cotransfected into cells. Relative promoter activity was determined 24 h posttransfection as the luciferase activity normalized for transfection efficiency using the β-galactosidase activity. Values are means and SD (n = 3). (Right) Percent repression by PRH for each reporter construct. Cells were transfected and reporter assays were carried out as described above except that cells were cotransfected with 1 μg pMUG1-Myc-PRH. Percent repression is promoter activity in the presence of PRH as a percentage of reporter activity in the absence of PRH. (B) K562 cells were transfected with PRH shRNA (sh) or control shRNA and grown in selection for 10 days. The cells were then retransfected with 5 μg of pSV-lacZ and 5 μg of the minimal TK reporter (TKmin; bars 1 and 3) or the minimal TK reporter containing PRH binding sites (TKPRH; bars 2 and 4). Twenty-four hours posttransfection, relative promoter activity was determined as described in the text. Values are means and SD (n = 3). (C) Cells were transfected and assayed for reporter activity as for panel A. The graph shows the activity of the R1 1.2, R2 1.3, and R2 0.4 reporters cotransfected into cells with 1 μg pMUG1-PRH, 1 μg pMUG1-PRH N187A, or 3 μg pCS2-Hex-VP16. (D) Cells were transfected and assayed for reporter activity as for panel A. The graph shows the activity of the A 2.9 and A 4.0 Vegf reporters with 3 μg or 6 μg pCS2-Hex-VP16.

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Construct, shRNA, Selection, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH KD cells are sensitive to VEGF inhibition. (A) K562 cells were transfected with SVC shRNA (sh) or PRH shRNA and then incubated with 50 μg/ml anti-VEGF antibody (bars 2 and 4) or an equal volume of DMSO (bars 1 and 3) for 72 h. An MTT assay was then used to calculate cell numbers. Values are means and SD (n = 5). (B) As for panel A except that cells were incubated with 50 ng/ml VEGF. Values are means and SD (n = 5). (C) As for panel A except that cells were incubated with DMSO (bars 1 and 4), 2 μM SU11248 (bars 2 and 5), or 2 μM SU1498 (bars 3 and 6). Values are means and SD (n = 3). (D) K562 cells transfected with SVC shRNA (black bars) or PRH shRNA (gray bars) or not shRNA transfected (white bars) were then transfected with a vector expressing PRH (3 μg) (bars 5 and 6) and/or vectors expressing VEGF, VEGFR-1, and VEGFR-2 (bars 2, 4, and 6) (total, 3 μg). Twenty-four hours posttransfection the cells were dual stained with PI/AV antibody for analysis by flow cytometry. The graph shows the means and SD (n = 3).

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Inhibition, Transfection, shRNA, Incubation, MTT Assay, Plasmid Preparation, Expressing, Staining, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH represses VSP genes in MCF-7 cells. (A) Western analysis of whole-cell extracts from untransfected MCF-7 cells or cells cotransfected with SVC shRNA or PRH shRNA (1+2). Extracts were stained with rabbit PRH antisera (top) and tubulin antibody (bottom). (B) Growth curves for MCF-7 cells transfected with SVC shRNA (sh) or PRH shRNA (1+2). Cells were selected with puromycin 24 h posttransfection, and after 5 days in selection, MTT assays were performed over 3 days. Results shown are representative of the results from 6 independent experiments. (C) Vegf, Vegfr-1, and Vegfr-2 mRNA levels in MCF-7 cells after shRNA cotransfection (as above). Levels of mRNA were determined by qRT-PCR using specific primers and compared to that for GAPDH. Black bars represent the SVC shRNA-targeted cells and gray bars the PRH shRNA (1+2)-targeted cells. Values are means and SD (n = 5). (D) Vegfr-1, Vegfr-2, and Vegf mRNA levels in MCF-7 cells 48 h after transfection with pMUG1 (empty vector) or pMUG1-Myc-PRH. mRNA levels were determined as above. Values are means and SD (n = 3).

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Western Blot, shRNA, Staining, Transfection, Selection, Cotransfection, Quantitative RT-PCR, Plasmid Preparation

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH represses VSP genes. (A) Western blot of whole-cell extracts from K562 cells transiently transfected with increasing amounts of a plasmid expressing Myc-tagged PRH. Proteins were stained with a mouse anti-PRH antibody (top), the Myc9E10 monoclonal antibody (middle), and a lamin A/C antibody (bottom). (B) Vegfr-1 and Vegf mRNA levels in K562 cells 48 h after transfection with 10 μg pMUG1 or pMUG1-Myc-PRH. mRNA levels were determined by qRT-PCR using specific primers and compared to those of GAPDH. Values are means and standard deviations (SD) (n = 5). (C) Western blot of whole-cell extracts from K562 cells cotransfected with shRNA plasmids SVC shRNA, PRH shRNA 1, PRH shRNA 2, and PRH shRNA (1+2) (4). Proteins were stained with rabbit PRH antibody and lamin A/C antibody. (D) Prh, Vegf, Vegfr-1, and Vegfr-2 mRNA levels after shRNA (sh) cotransfection. Black bars represent SVC shRNA-targeted cells and gray bars PRH shRNA (1+2)-targeted cells. Values are means and SD (n = 5). (E) Western blot of whole-cell extract from K562 cells transfected with empty pMUG1 or pMUG1-MycPRH. Proteins were stained with VEGF antibody or lamin A/C antibody.

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Staining, Quantitative RT-PCR, shRNA, Cotransfection

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH binds to the Vegf, Vegfr-1, and Vegfr-2 promoters in cells. (A) Schematic of the human Vegfr-1 promoter (top line), with the positions of potential PRH binding sites indicated as bars. C1 to C5 indicate clusters of potential sites. The arrow corresponds to the first exon. Each inset panel shows the results of PCRs using the following templates: (1) input DNA, (2) DNA precipitated from cells expressing Myc-PRH with beads plus Myc antibody, (3) DNA precipitated from cells expressing Myc-PRH with beads plus normal IgG. The amplicons are indicated by solid bars. In the same experiment, primers within the actin promoter did not produce a product (left inset). (B) As for panel A except that the schematic and ChIP results are for the Vegfr-2 promoter. (C) As for panel A except that the schematic and ChIP results are for the Vegf promoter.

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Binding Assay, Expressing

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH directly represses the Vegf, Vegfr-1, and Vegfr-2 promoters. (A, left) Schematic representations of the Vegfr-1, Vegfr-2, and Vegf promoters indicating the position of the first exon for each gene (white arrows) and the promoter derivatives (black arrows) used in this study. (Middle) Relative promoter activity found in K562 cell extracts following transfection with Vegfr-1, Vegfr-2, or Vegf reporters. Five micrograms of each reporter and 5 μg of the β-galactosidase plasmid (pSV-lacZ) were cotransfected into cells. Relative promoter activity was determined 24 h posttransfection as the luciferase activity normalized for transfection efficiency using the β-galactosidase activity. Values are means and SD (n = 3). (Right) Percent repression by PRH for each reporter construct. Cells were transfected and reporter assays were carried out as described above except that cells were cotransfected with 1 μg pMUG1-Myc-PRH. Percent repression is promoter activity in the presence of PRH as a percentage of reporter activity in the absence of PRH. (B) K562 cells were transfected with PRH shRNA (sh) or control shRNA and grown in selection for 10 days. The cells were then retransfected with 5 μg of pSV-lacZ and 5 μg of the minimal TK reporter (TKmin; bars 1 and 3) or the minimal TK reporter containing PRH binding sites (TKPRH; bars 2 and 4). Twenty-four hours posttransfection, relative promoter activity was determined as described in the text. Values are means and SD (n = 3). (C) Cells were transfected and assayed for reporter activity as for panel A. The graph shows the activity of the R1 1.2, R2 1.3, and R2 0.4 reporters cotransfected into cells with 1 μg pMUG1-PRH, 1 μg pMUG1-PRH N187A, or 3 μg pCS2-Hex-VP16. (D) Cells were transfected and assayed for reporter activity as for panel A. The graph shows the activity of the A 2.9 and A 4.0 Vegf reporters with 3 μg or 6 μg pCS2-Hex-VP16.

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Construct, shRNA, Selection, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH KD cells are sensitive to VEGF inhibition. (A) K562 cells were transfected with SVC shRNA (sh) or PRH shRNA and then incubated with 50 μg/ml anti-VEGF antibody (bars 2 and 4) or an equal volume of DMSO (bars 1 and 3) for 72 h. An MTT assay was then used to calculate cell numbers. Values are means and SD (n = 5). (B) As for panel A except that cells were incubated with 50 ng/ml VEGF. Values are means and SD (n = 5). (C) As for panel A except that cells were incubated with DMSO (bars 1 and 4), 2 μM SU11248 (bars 2 and 5), or 2 μM SU1498 (bars 3 and 6). Values are means and SD (n = 3). (D) K562 cells transfected with SVC shRNA (black bars) or PRH shRNA (gray bars) or not shRNA transfected (white bars) were then transfected with a vector expressing PRH (3 μg) (bars 5 and 6) and/or vectors expressing VEGF, VEGFR-1, and VEGFR-2 (bars 2, 4, and 6) (total, 3 μg). Twenty-four hours posttransfection the cells were dual stained with PI/AV antibody for analysis by flow cytometry. The graph shows the means and SD (n = 3).

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Inhibition, Transfection, shRNA, Incubation, MTT Assay, Plasmid Preparation, Expressing, Staining, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: PRH/Hhex Controls Cell Survival through Coordinate Transcriptional Regulation of Vascular Endothelial Growth Factor Signaling ▿

doi: 10.1128/MCB.01511-09

Figure Lengend Snippet: PRH represses VSP genes in MCF-7 cells. (A) Western analysis of whole-cell extracts from untransfected MCF-7 cells or cells cotransfected with SVC shRNA or PRH shRNA (1+2). Extracts were stained with rabbit PRH antisera (top) and tubulin antibody (bottom). (B) Growth curves for MCF-7 cells transfected with SVC shRNA (sh) or PRH shRNA (1+2). Cells were selected with puromycin 24 h posttransfection, and after 5 days in selection, MTT assays were performed over 3 days. Results shown are representative of the results from 6 independent experiments. (C) Vegf, Vegfr-1, and Vegfr-2 mRNA levels in MCF-7 cells after shRNA cotransfection (as above). Levels of mRNA were determined by qRT-PCR using specific primers and compared to that for GAPDH. Black bars represent the SVC shRNA-targeted cells and gray bars the PRH shRNA (1+2)-targeted cells. Values are means and SD (n = 5). (D) Vegfr-1, Vegfr-2, and Vegf mRNA levels in MCF-7 cells 48 h after transfection with pMUG1 (empty vector) or pMUG1-Myc-PRH. mRNA levels were determined as above. Values are means and SD (n = 3).

Article Snippet: PRH short hairpin RNA (shRNA) plasmids and green fluorescent protein (GFP) shRNA were from Origene. pBJ Flt-1, pBJ KDR, and pCDNA3 VEGF 165 are the expression plasmids for VEGFR-1, VEGFR-2 and VEGF, respectively, and were a kind gift from D. Bates.

Techniques: Western Blot, shRNA, Staining, Transfection, Selection, Cotransfection, Quantitative RT-PCR, Plasmid Preparation