p53 responsive luciferase reporter vector  (Addgene inc)

Journal: Journal of Hematology & Oncology

Article Title: ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21

doi: 10.1186/s13045-020-00943-w

Figure Lengend Snippet: ETV4 downregulates CDKN1A promoter regardless of ETV4-binding sites. a Diagram of the human CDKN1A promoter (top) and of the vectors (BS-AB) in which firefly luciferase expression is driven by the 1656 bp region upstream the CDKN1A TSS (bottom). ETV4 BSs are indicated by a diamond and p53 BSs are indicated by a circle. Four different variants of the BS-AB vector have been used. The BS-AB WT with both wild-type ETV4 BS-A and BS-B. In the other 3 vectors, one or both of this ETV4 BS have been mutated; the mutated sites are in black the normal sites are in white. b Quantification of dual luciferase reporter assay in PC3 cells transiently transfected with the indicated four BS-AB vectors (see above). The bar diagram shows the relative luciferase activity (Firefly/Renilla ratio) from cells transduced with one of 2 anti-ETV4 shRNA (shETV4a and shETV4b) normalized to those transduced with an irrelevant shRNA (CTL). Data represent average and standard error of triplicate measurement of 3 independent experiments. c Quantification of dual luciferase reporter assay in RWPE cells transiently transfected with the indicated four BS-AB vectors (see above). The bar diagram shows the relative luciferase activity (Firefly/Renilla ratio) from cells transfected with an ETV4-expressing vector (+ETV4) normalized to those transfected with an empty vector (CTL). Data represent average and standard error of triplicate measurement of 3 independent experiments. * P ≤ 0.05, ** P ≤ 0.01

Article Snippet: Derivative vectors were obtained mutating the putative ETV4-binding sites with the QuikChange II site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA): the normal sequence “CCGGAAGC” of the ETV4 BS A (Figs. and a) was replaced with the mutated sequence “CCGATATC”; the normal sequence “AGAGGAAGAA” of ETV4 BS-B (Figs. and a) was replaced with the mutated sequence “AACCGAAGAA.” In addition, also a p53-responsive luciferase reporter vector (Addgene) [ ] was used.

Techniques: Binding Assay, Luciferase, Expressing, Plasmid Preparation, Reporter Assay, Transfection, Activity Assay, Transduction, shRNA

Journal: Journal of Hematology & Oncology

Article Title: ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21

doi: 10.1186/s13045-020-00943-w

Figure Lengend Snippet: ETV4 downregulates the CDKN1A promoter also through the modulation of p53 levels. a Dual luciferase reporter assay in PNT1A and RWPE cells transiently transfected with a vector containing firefly luciferase downstream the 20 bp human CDKN1A p53-binding site (p53 BS) (top panel). The bar diagram shows the relative luciferase activity (Firefly/Renilla ratio) from cells transfected with either an ETV4-expressing vector (+ETV4, grey bars) or a p53-expressing vector (+p53, striped bars), and normalized to those transfected with an empty vector (CTL, black bar). Data represent average and standard error of triplicate measurements of 3 independent experiments. b Representative western blots of p53 in human RWPE cells transfected with either an empty vector (CTL) or an ETV4-expressing vector (+ETV4). Beta actin is used as loading control. c Bar diagram of p53 protein levels measured by western blot analysis in human RWPE cells transfected with an ETV4-expressing vector (+ETV4) normalized to those transfected with an empty vector (CTL). Average and standard error values of the relative quantification are shown. Seven independent experiments have been performed. d Representative western blots of p53 protein in the 3 prostate lobes of wild-type (WT) and of ETV4 transgenic mice (lines ETV4 A and ETV4 B); beta-actin was used as loading control. The relative densitometric quantifications are shown for each lane. e Bar diagram of p53 protein levels measured by western blot analysis in the 3 prostate lobes of ETV4 transgenic mice (lines ETV4 A and ETV4 B) normalized to wild-type mice (WT). Average and standard error values of the relative quantification are shown; n : number of mice. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

Article Snippet: Derivative vectors were obtained mutating the putative ETV4-binding sites with the QuikChange II site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA): the normal sequence “CCGGAAGC” of the ETV4 BS A (Figs. and a) was replaced with the mutated sequence “CCGATATC”; the normal sequence “AGAGGAAGAA” of ETV4 BS-B (Figs. and a) was replaced with the mutated sequence “AACCGAAGAA.” In addition, also a p53-responsive luciferase reporter vector (Addgene) [ ] was used.

Techniques: Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Binding Assay, Activity Assay, Expressing, Western Blot, Control, Quantitative Proteomics, Transgenic Assay

Journal: Cell Cycle

Article Title: The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G 1 arrest

doi: 10.4161/cc.11.2.18999

Figure Lengend Snippet: RAX/PACT enhances the association of p53 with Ubc9 to promote p53 sumoylation at lysine 386. (A) RAX/PACT expression promotes the association of p53 and Ubc9. Ubc9, p53 and Flag-tagged RAX/PACT were transiently transfected into H1299 cells. Immunoprecipitation was performed with anti-Ubc9 antibody, and co-immunoprecipitation was detected by western blotting. (B) RAX/PACT enhances p53 sumoylation in vivo. His-tagged SUMO-1 was co-transfected with p53 and the indicated expression plasmids into H1299 cells. Affinity chromatography was used to isolate His-SUMO-1 conjugated proteins, and the sumoylated p53 population was detected by western blotting with antibody to p53. Co-expression of MDM2 and ARF synergize with RAX/PACT to further enhance p53 sumoylation. (C) RAX/PACT mediates p53 sumoylation at lysine 386 since RAX/PACT does not promote sumoylation of the p53(K386R) mutant. (D) RAX/PACT-induced p53 sumoylation is reversible. Co-expression of the SUMO-specific protease SENP1 inhibits RAX/PACT-induced sumoylation of p53. (E) The expression plasmids for p53-Ubc9, p53 (K386R)-Ubc9, p53-Ubc9 (C93S) as well as HA-SUMO-1 and Flag-RAX were co-transfected with 0.1 µg of GFP in H1299 cells. SUMO-conjugated or non-conjugated p53 were detected by western blot. GFP was used as a transfection efficiency control.

Article Snippet: The ratio of p53 reporter/Renilla reporter activity was used as an indicator of p53 transcription activity. p53-TA (containing the wt p53 response elements) luciferase reporter vectors were obtained from BD Clontech.

Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, In Vivo, Affinity Chromatography, Mutagenesis

Journal: Cell Cycle

Article Title: The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G 1 arrest

doi: 10.4161/cc.11.2.18999

Figure Lengend Snippet: RAX/PACT increases the activity of p53. RAX/PACT promotes p53-dependent transcription in a dose-dependent manner. (A) H1299 cells (p53-null) and (B) U2OS cells expressing endogenous p53 were transiently transfected with a p53-inducible firefly luciferase plasmid, Renilla luciferase control plasmid and 0.1, 0.3 or 0.6 µg of the indicated expression constructs. Twenty-four hours after transfection, cells were lysed, and the firefly/Renilla luciferase ratio was measured by dual luciferase assay. The average of at least three similar dual luciferase assays and a representative western blot are shown. (C) Cell cycle analysis of H1299 cells 24 h after transfection with plasmids expressing either GFP alone, RAX/PACT and/or p53 as indicated. (D) U2OS cells stably expressing siRNA to RAX/PACT display a > 90% reduction in RAX/PACT protein by western blot. (E) Quantitative real-time PCR (qPCR) to measure gene expression of RAX/PACT, p53, MDM2 and the p53 targets p21, PUMA and BAX in U2OS cells stably expressing either siRNA to RAX/PACT or control siRNA.

Article Snippet: The ratio of p53 reporter/Renilla reporter activity was used as an indicator of p53 transcription activity. p53-TA (containing the wt p53 response elements) luciferase reporter vectors were obtained from BD Clontech.

Techniques: Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Construct, Western Blot, Cell Cycle Assay, Stable Transfection, Real-time Polymerase Chain Reaction

Journal: Cell Cycle

Article Title: The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G 1 arrest

doi: 10.4161/cc.11.2.18999

Figure Lengend Snippet: RAX/PACT-dependent G 1 cell cycle arrest is dependent on p53 sumoylation

Article Snippet: The ratio of p53 reporter/Renilla reporter activity was used as an indicator of p53 transcription activity. p53-TA (containing the wt p53 response elements) luciferase reporter vectors were obtained from BD Clontech.

Techniques:

Journal: Cell Cycle

Article Title: The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G 1 arrest

doi: 10.4161/cc.11.2.18999

Figure Lengend Snippet: PKR promotes p53 activity. (A) Co-expression of PKR and p53 promotes G1 cell cycle arrest. Cell cycle analysis of H1299 cells 24 h after transfection with plasmids expressing either GFP alone, PKR and/or p53 as indicated. (B) Exogenous RAX/PACT expression increases the level of p53 and p21 in normal pkr+/+ MEF cells but not pkr−/− MEF cells. RAX/PACT expression and endogenous levels of PKR, p53, p21 and actin were determined by western blot using lysate of MEF cells from pkr−/− mice or paired pkr+/+ littermates stably expressing either vector control or Flag-RAX/PACT. (C) Left part: p53 and p21 levels are increased following 10 Gy of gamma irradiation. Right part: following gamma irradiation, pkr−/− MEF cells or pkr−/− MEF stably expressing Flag-RAX/PACT fail to upregulate p53 and p21 expression.

Article Snippet: The ratio of p53 reporter/Renilla reporter activity was used as an indicator of p53 transcription activity. p53-TA (containing the wt p53 response elements) luciferase reporter vectors were obtained from BD Clontech.

Techniques: Activity Assay, Expressing, Cell Cycle Assay, Transfection, Western Blot, Stable Transfection, Plasmid Preparation, Irradiation

Journal: Cell Cycle

Article Title: The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G 1 arrest

doi: 10.4161/cc.11.2.18999

Figure Lengend Snippet: PKR synergizes with p53 to promote G 1 cell cycle arrest

Article Snippet: The ratio of p53 reporter/Renilla reporter activity was used as an indicator of p53 transcription activity. p53-TA (containing the wt p53 response elements) luciferase reporter vectors were obtained from BD Clontech.

Techniques:

Journal: Cell Cycle

Article Title: The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G 1 arrest

doi: 10.4161/cc.11.2.18999

Figure Lengend Snippet: RAX/PACT-PKR promotes p53 stability following stress and p53 phosphorylation on serine 392. (A) PKR expression is reduced by > 80% in U2OS cells by siRNA knockdown. (B) The half-life of p53 is decreased in U2OS cells with reduced PKR following treatment with 5 µM doxorubicin. (C) RAX/PACT expression stimulates p53 phosphorylation at serine 392 when co-expressed with wild-type p53 but not mutant p53(K386R).

Article Snippet: The ratio of p53 reporter/Renilla reporter activity was used as an indicator of p53 transcription activity. p53-TA (containing the wt p53 response elements) luciferase reporter vectors were obtained from BD Clontech.

Techniques: Expressing, Mutagenesis