p53 responsive luciferase reporter pg13 luc  (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: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: MAGEA3 interacts with KAP1 and inhibits p53 transcriptional activity. A. Co-IP assays were performed in HeLa cells and SiHa cells cotransfected with Lv-MAGEA3 and pcDNA3.1-KAP1. Cell lysates were immunoprecipitated (IP) with control IgG or the indicated antibody, and the precipitated protein was detected by immunoblotting (IB) analysis with the indicated antibody. Cell extracts were used as a positive control (Input). B. Effects of MAGEA3 or KAP1 on p53 activity, as indicated by the reporter plasmid pp53-TA-luc. HeLa cells (with endogenous wild-type p53) grown on a 12-well plate were cotransfected with the pp53-TA-luc and the indicated plasmids. Luciferase was measured at 48 h posttransfection and normalized according to Renilla luciferase activities. Data (means ± SD) are represented as fold differences relative to that observed in cells without transfecting the indicated plasmids. C, D. Western blotting analysis of the p53 and KAP1 proteins following overexpression or knockdown of MAGEA3 in SiHa (Ca, Cb) and HeLa (Da, Db) cells. *P<0.05, **P<0.01 vs. control groups. E. Immunohistochemical analysis of p53 expression in the Lv-MAGEA3 group and Lv-NC group of xenograft tumors. Bar length: 100 µm.

Article Snippet: Dual-luciferase reporter gene assay Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Positive Control, Plasmid Preparation, Luciferase, Over Expression, Knockdown, Immunohistochemical staining, Expressing

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: Effects of MAGEA3 expression on the protein levels of p53 downstream targets. A, B. Western blotting analysis of p53, p21, Bax, Bcl2, PUMA, cyclin D1, and cleaved caspase-3 proteins following overexpression or knockdown of MAGEA3 in SiHa (Aa, Ab) and HeLa (Ba, Bb) cells. *P<0.05, **P<0.01 vs. control groups. C. Immunohistochemical analysis of p21 and Bax expression in the Lv-MAGEA3 group and Lv-NC group of xenograft tumors. Bar length: 100 µm.

Article Snippet: Dual-luciferase reporter gene assay Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Expressing, Western Blot, Over Expression, Knockdown, Control, Immunohistochemical staining

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: Nutlin3 reverses the effects of MAGEA3 overexpression on the p53 signaling pathway, cell cycle, and apoptosis. A. Flow cytometric analysis of the cell cycle and apoptosis after treatment with Nutlin3 (10 μM) for 48 h. B. Western blotting analysis of the indicated proteins after treatment with Nutli3 (10 μM) for 48 h. *P<0.05, **P<0.01 vs. Lv-NC; ##P<0.01 vs. Lv-MAGEA3.

Article Snippet: Dual-luciferase reporter gene assay Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Over Expression, Western Blot

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: MAGEA3 interacts with KAP1 and inhibits p53 transcriptional activity. A. Co-IP assays were performed in HeLa cells and SiHa cells cotransfected with Lv-MAGEA3 and pcDNA3.1-KAP1. Cell lysates were immunoprecipitated (IP) with control IgG or the indicated antibody, and the precipitated protein was detected by immunoblotting (IB) analysis with the indicated antibody. Cell extracts were used as a positive control (Input). B. Effects of MAGEA3 or KAP1 on p53 activity, as indicated by the reporter plasmid pp53-TA-luc. HeLa cells (with endogenous wild-type p53) grown on a 12-well plate were cotransfected with the pp53-TA-luc and the indicated plasmids. Luciferase was measured at 48 h posttransfection and normalized according to Renilla luciferase activities. Data (means ± SD) are represented as fold differences relative to that observed in cells without transfecting the indicated plasmids. C, D. Western blotting analysis of the p53 and KAP1 proteins following overexpression or knockdown of MAGEA3 in SiHa (Ca, Cb) and HeLa (Da, Db) cells. *P<0.05, **P<0.01 vs. control groups. E. Immunohistochemical analysis of p53 expression in the Lv-MAGEA3 group and Lv-NC group of xenograft tumors. Bar length: 100 µm.

Article Snippet: Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Western Blot, Positive Control, Plasmid Preparation, Luciferase, Over Expression, Knockdown, Immunohistochemical staining, Expressing

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: Effects of MAGEA3 expression on the protein levels of p53 downstream targets. A, B. Western blotting analysis of p53, p21, Bax, Bcl2, PUMA, cyclin D1, and cleaved caspase-3 proteins following overexpression or knockdown of MAGEA3 in SiHa (Aa, Ab) and HeLa (Ba, Bb) cells. *P<0.05, **P<0.01 vs. control groups. C. Immunohistochemical analysis of p21 and Bax expression in the Lv-MAGEA3 group and Lv-NC group of xenograft tumors. Bar length: 100 µm.

Article Snippet: Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Expressing, Western Blot, Over Expression, Knockdown, Control, Immunohistochemical staining

Journal: American Journal of Translational Research

Article Title: MAGEA3 promotes proliferation and suppresses apoptosis in cervical cancer cells by inhibiting the KAP1/p53 signaling pathway

doi:

Figure Lengend Snippet: Nutlin3 reverses the effects of MAGEA3 overexpression on the p53 signaling pathway, cell cycle, and apoptosis. A. Flow cytometric analysis of the cell cycle and apoptosis after treatment with Nutlin3 (10 μM) for 48 h. B. Western blotting analysis of the indicated proteins after treatment with Nutli3 (10 μM) for 48 h. *P<0.05, **P<0.01 vs. Lv-NC; ##P<0.01 vs. Lv-MAGEA3.

Article Snippet: Cells were cultured at a density of 5×10 4 cells/well in 12-well plates and cotransfected with the p53-responsive luciferase reporter plasmid (pp53-TA-luc, containing p53 response element, Beyotime, Shanghai, China) along with the Renilla luciferase reporter plasmid (pGMR-TK, Genomeditech, Shanghai, China) for 6 h. Some cells were further transfected with the indicated plasmids or lentivirus.

Techniques: Over Expression, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: TFF3 overexpression enhances p53 transcriptional activity and protein expression and increases miR-34a expression with downstream reduction of EMP1 in Y79 RB cells. ( A ) Quantitative Real-time PCR confirmation of TFF3 lentiviral overexpression (Trefoil factor family peptide 3 (TFF3)) in Y79 cells compared to control cells (ctr). ( B ) Luciferase assays were performed with Y79 cells transiently transfected with TFF3 or empty vector control (ctr) in addition to wild-type PG13-Luc (wt PG13) or mutant MG15-Luc (mut MG15). Forced TFF3 expression leads to an increased luciferase signal upon p53 promotor activation in Y79 cells. ( C ) Western blot analysis showing increased p53 and TFF3 protein levels after TFF3 overexpression (TFF3). The indicated intensity ratios of p53 and TFF3 protein levels relative to β-actin levels were calculated using ImageJ software. ( D , E ) Quantitative real-time PCR analysis of miR-34a and EMP1 expression levels in Y79 cells compared to control cells after lentiviral TFF3 overexpression (ctr). Values are means of at least 3 independent experiments ± SEM. * p -value < 0.05, ** p -value < 0.01; statistical differences compared to the control group calculated by Student’s t -test or one-way ANOVA and Newman-Keuls post test.

Article Snippet: TFF3 dependent p53 activity was measured with the p53 responsive reporter PG13- Luc (with p53 binding site) and the non-responsive reporter MG15- Luc (with a mutant p53 binding site; Addgene [ ]) after co-transfection of pCS2+_UBQ_TFF3 or an empty control vector (pCS2+_UBQ).

Techniques: Over Expression, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot, Software

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: TFF3 overexpression enhances p53 transcriptional activity and protein expression and increases miR-34a expression with downstream reduction of EMP1 in Y79 RB cells. ( A ) Quantitative Real-time PCR confirmation of TFF3 lentiviral overexpression (Trefoil factor family peptide 3 (TFF3)) in Y79 cells compared to control cells (ctr). ( B ) Luciferase assays were performed with Y79 cells transiently transfected with TFF3 or empty vector control (ctr) in addition to wild-type PG13-Luc (wt PG13) or mutant MG15-Luc (mut MG15). Forced TFF3 expression leads to an increased luciferase signal upon p53 promotor activation in Y79 cells. ( C ) Western blot analysis showing increased p53 and TFF3 protein levels after TFF3 overexpression (TFF3). The indicated intensity ratios of p53 and TFF3 protein levels relative to β-actin levels were calculated using ImageJ software. ( D , E ) Quantitative real-time PCR analysis of miR-34a and EMP1 expression levels in Y79 cells compared to control cells after lentiviral TFF3 overexpression (ctr). Values are means of at least 3 independent experiments ± SEM. * p -value < 0.05, ** p -value < 0.01; statistical differences compared to the control group calculated by Student’s t -test or one-way ANOVA and Newman-Keuls post test.

Article Snippet: TFF3 dependent p53 activity was measured with the p53 responsive reporter PG13- Luc (with p53 binding site) and the non-responsive reporter MG15- Luc (with a mutant p53 binding site; Addgene [ ]) after co-transfection of pCS2+_UBQ_TFF3 or an empty control vector (pCS2+_UBQ).

Techniques: Over Expression, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot, Software