Journal: Nature cell biology

Article Title: TAp73 enhances the pentose phosphate pathway and supports cell proliferation

doi: 10.1038/ncb2789

Figure Lengend Snippet: p73 regulates PPP flux, G6PD activity and expression ( a , b ) TAp73 +/+ and TAp73 −/− MEF cells ( a ), or Δ Np73 +/+ and Δ Np73 −/− MEF cells ( b ), were cultured in medium containing [2- 13 C]glucose. Oxidative PPP flux was measured based on the rate of glucose consumption and the ratio of 13 C incorporated into carbon 3 (indicating PPP) and carbon 2 (indicating glycolysis) of lactate determined by NMR spectroscopy. Data are means ± SD (n = 3 independent experiments). ( c , d ) Top: G6PD activity in two independent clones of TAp73 −/− MEFs ( c ), Δ Np73 −/− MEFs ( d ), and the corresponding wild-type MEFs. Bottom: G6PD and β-actin mRNA levels, with the relative ratios of G6PD to β-actin indicated. Data are means ± SD (n = 3 independent experiments). Western blots represent two independent experiments. ( e-i ) G6PD activity in U2OS cells ( e ), isogenic p53 +/+ ( f ), p53 −/− ( g ), or p21 −/− ( h ) HCT116 cells, or IMR90 cells ( i ) that were treated with either control (−) or p73 siRNA. The expression of G6PD, TAp73, and actin was detected using Western blot (WB) ( e-h ) or RT-PCR ( f ). Data are means ± SD (n = 3 independent experiments). Western blots represent three independent experiments.

Article Snippet: Antibodies against the following proteins/epitopes were used in this study with the company, catalog number, and dilution or concentration indicated: GFP (Clontech Laboratories, Mountain View, CA; 632381; 1:4,000), actin (Sigma-Aldrich, St Louis, MO; A2066; 1:4,000), Flag (Sigma, F3165, 1:4,000), p53 (DO-1) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126 HRP; 1:1,000), p73 (Imgenex Corp, San Diego, CA; IMG-259A; 1:1,000) (Bethyl Laboratories, Montgomery, TX; A300-126A; 1:1,000) (Sigma, SAB4300354, 1:1,000), mouse p73 (Santa Cruz, sc-7238, 1:500) (Cell Signaling Technology, Danvers, MA; 4300354; 1:1,000), G6PD (Sigma, HPA000834, 1:3,000), p21 (Cell Signaling, 2947, 1:1,000), and fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (BD Bioscience Pharmingen; 556028; 10 μM).

Techniques: Activity Assay, Expressing, Cell Culture, Spectroscopy, Clone Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Nature cell biology

Article Title: TAp73 enhances the pentose phosphate pathway and supports cell proliferation

doi: 10.1038/ncb2789

Figure Lengend Snippet: p73 regulates G6PD transcription ( a ) mRNA levels in U2OS cells that were stably infected with lentiviruses expressing a control (Ctrl) shRNA or shRNA against the indicated p53 family gene. ( b ) IMR90 cells were infected with lentiviruses expressing a control shRNA or a TAp73 -specific shRNA for indicated durations. Protein and mRNA expression was detected using RT-PCR and WB, respectively. Data represent three independent experiments. ( c ) H1299 cells were transfected with increasing amounts of control plasmid or plasmid expressing the indicated p53 family protein. Western blots represent two independent experiments, and the relative ratios of G6PD to β-actin are given. G6PD mRNA levels were detected by quantitative RT-PCR . ( d ) TAp73 +/+ and TAp73 −/− MEF cells were treated with ETP (20 μM) for the indicated durations and were analyzed by RT-PCR and WB. ( e ) IMR90 cells transfected with p73 siRNA or control siRNA were treated with increasing amounts of ETP. G6PD expression was analyzed by RT-PCR and WB. ( f ) Schematic representation of human G6PD genomic structure. Shown are the exon/intron organization, consensus p53 family protein RE, the potential p73 RE within the first intron, and the corresponding mutant p73 RE (with mutated nucleotides underlined). Arrows mark the positions of the primers used for PCR in the ChIP assay. UTR: un-translated region. ( g ) A549 cells that were treated with and without doxorubicin (DOX, 1 μg/ml) were analyzed by ChIP assay with the indicated antibodies. Data represent two independent experiments. ( h ) H1299 cells transfected with indicated GFP plasmids were analyzed by ChIP assay using anti-GFP antibody or normal mouse IgG and G6PD specific primers. The DNA size standard (in base pairs) is shown on the left. Data represent two independent experiments. ( i ) Binding of endogenous p53, p63, and p73 to the puma genomic locus in A549 cells was analyzed by ChIP assay. Anti-p21 antibody and no antibody (−) were used as controls. ( j , k ) Luciferase reporter constructs containing the wild-type ( j ) or mutant ( k ) p73 RE were transfected into H1299 cells together with increasing amounts of indicated plasmids. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data are means ± SD (n = 3 independent experiments). ( l ) Protein expression of G6PD, TAp73, p53 and p21 in MCF-7 (wild-type p53), SK-BR-3 (p53 R175H), and MDA-MB-468 (p53 R273H) cells treated with p53 or control siRNA. Western blots represent three independent experiments.

Article Snippet: Antibodies against the following proteins/epitopes were used in this study with the company, catalog number, and dilution or concentration indicated: GFP (Clontech Laboratories, Mountain View, CA; 632381; 1:4,000), actin (Sigma-Aldrich, St Louis, MO; A2066; 1:4,000), Flag (Sigma, F3165, 1:4,000), p53 (DO-1) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126 HRP; 1:1,000), p73 (Imgenex Corp, San Diego, CA; IMG-259A; 1:1,000) (Bethyl Laboratories, Montgomery, TX; A300-126A; 1:1,000) (Sigma, SAB4300354, 1:1,000), mouse p73 (Santa Cruz, sc-7238, 1:500) (Cell Signaling Technology, Danvers, MA; 4300354; 1:1,000), G6PD (Sigma, HPA000834, 1:3,000), p21 (Cell Signaling, 2947, 1:1,000), and fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (BD Bioscience Pharmingen; 556028; 10 μM).

Techniques: Stable Transfection, Infection, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Mutagenesis, Binding Assay, Luciferase, Construct

Journal: Nature cell biology

Article Title: TAp73 enhances the pentose phosphate pathway and supports cell proliferation

doi: 10.1038/ncb2789

Figure Lengend Snippet: p73 regulates NADPH homeostasis. ( a , b ) NADPH levels ( a ) and NADP + /NADPH ratios ( b ) in two independent clones of TAp73 +/+ and TAp73 −/− MEF cells are shown. Data are means ± SD (n = 3 independent experiments). ( c , d ) NADPH levels ( c ) and NADP + /NADPH ratios ( d ) in U2OS cells transfected with control (−) or p73 siRNA. Data are means ± SD (n = 3 independent experiments). ( e,f ) G6PD activity ( e ) and NADP + /NADPH ratios ( f ) in TAp73 +/+ and TAp73 −/− MEF cells stably overexpressing G6PD or vector control are shown. Protein expression was analyzed ( e ). Data are means ± SD (n = 3 independent experiments). Western blots represent three independent experiments. ( g,h ) G6PD activity ( g ) and NADP + /NADPH ratios ( h ) in U2OS cells stably overexpressing G6PD or vector control in the presence or absence of p73 siRNA are shown. Protein expression was analyzed ( g ). Data are means ± SD (n = 3 independent experiments). Western blots represent three independent experiments.

Article Snippet: Antibodies against the following proteins/epitopes were used in this study with the company, catalog number, and dilution or concentration indicated: GFP (Clontech Laboratories, Mountain View, CA; 632381; 1:4,000), actin (Sigma-Aldrich, St Louis, MO; A2066; 1:4,000), Flag (Sigma, F3165, 1:4,000), p53 (DO-1) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126 HRP; 1:1,000), p73 (Imgenex Corp, San Diego, CA; IMG-259A; 1:1,000) (Bethyl Laboratories, Montgomery, TX; A300-126A; 1:1,000) (Sigma, SAB4300354, 1:1,000), mouse p73 (Santa Cruz, sc-7238, 1:500) (Cell Signaling Technology, Danvers, MA; 4300354; 1:1,000), G6PD (Sigma, HPA000834, 1:3,000), p21 (Cell Signaling, 2947, 1:1,000), and fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (BD Bioscience Pharmingen; 556028; 10 μM).

Techniques: Clone Assay, Transfection, Activity Assay, Stable Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Nature cell biology

Article Title: TAp73 enhances the pentose phosphate pathway and supports cell proliferation

doi: 10.1038/ncb2789

Figure Lengend Snippet: A role of p73 in regulating ROS ( a ) ROS levels in TAp73 +/+ and TAp73 −/− MEF cells treated with control or G6pd siRNA. Protein expression is shown (Right). Western blots represent three independent experiments. ( b ) ROS levels in U2OS cells transfected with the indicated siRNA. ( c ) TAp73 +/+ and TAp73 −/− MEF cells were treated with or without 150 μM H 2 O 2 for 1 h and then cultured for 24 h in the presence or absence of G6pd siRNA. Cell viability was analyzed by trypan blue staining. Data are means ± SD (n = 3 independent experiments). ( d ) ROS levels in TAp73 +/+ and TAp73 −/− MEF cells stably overexpressing G6PD or vector control. Protein expression is shown in Fig. 4 e . ( e ) U2OS cells stably overexpressing G6PD or vector control were transfected with p73 or control siRNA as indicated. ROS levels were analyzed. ( f ) U2OS cells stably overexpressing G6PD or vector control were transfected with control siRNA (−) or p73 siRNA as indicated. Cells were treated with or without 100 μM H 2 O 2 for 24 h and cell viability was analyzed by trypan blue staining. Data are means ± SD (n = 3 independent experiments).

Article Snippet: Antibodies against the following proteins/epitopes were used in this study with the company, catalog number, and dilution or concentration indicated: GFP (Clontech Laboratories, Mountain View, CA; 632381; 1:4,000), actin (Sigma-Aldrich, St Louis, MO; A2066; 1:4,000), Flag (Sigma, F3165, 1:4,000), p53 (DO-1) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126 HRP; 1:1,000), p73 (Imgenex Corp, San Diego, CA; IMG-259A; 1:1,000) (Bethyl Laboratories, Montgomery, TX; A300-126A; 1:1,000) (Sigma, SAB4300354, 1:1,000), mouse p73 (Santa Cruz, sc-7238, 1:500) (Cell Signaling Technology, Danvers, MA; 4300354; 1:1,000), G6PD (Sigma, HPA000834, 1:3,000), p21 (Cell Signaling, 2947, 1:1,000), and fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (BD Bioscience Pharmingen; 556028; 10 μM).

Techniques: Expressing, Western Blot, Transfection, Cell Culture, Staining, Stable Transfection, Plasmid Preparation

Journal: Nature cell biology

Article Title: TAp73 enhances the pentose phosphate pathway and supports cell proliferation

doi: 10.1038/ncb2789

Figure Lengend Snippet: p73 regulates DNA synthesis ( a ) TAp73 +/+ and TAp73 −/− MEF cells were treated with or without DHEA. DNA synthesis was measured by BrdU incorporation. Percentages of BrdU-positive cells are shown as mean ± SD (n = 3 independent experiments). ( b, c ) U2OS cells ( b ) and p53 +/+ HCT116 cells ( c ) were transfected with control siRNA (−), p73 siRNA, and G6PD siRNA as indicated. Cells were assayed for BrdU incorporation. Protein expression is shown below. Data are means ± SD (n = 3 independent experiments). Western blots represent three independent experiments. ( d ) Cell-cycle profile of p53 +/+ HCT116 cells transfected with p73 siRNA, G6PD siRNA, and control siRNA as indicated. Western blots represent three independent experiments. ( e ) U2OS cells were transfected with control siRNA, p73 siRNA, and Flag-G6PD as indicated. BrdU incorporation (top) and protein expression (bottom) are shown. Data are means ± SD (n = 3 independent experiments). Western blots represent three independent experiments. ( f ) Percentage of SA-β-gal positive cells in IMR90 cells stably overexpressing G6PD or vector control in the presence or absence of p73 siRNA. Data are means ± SD (n = 3 independent experiments). Western blots represent three independent experiments.

Article Snippet: Antibodies against the following proteins/epitopes were used in this study with the company, catalog number, and dilution or concentration indicated: GFP (Clontech Laboratories, Mountain View, CA; 632381; 1:4,000), actin (Sigma-Aldrich, St Louis, MO; A2066; 1:4,000), Flag (Sigma, F3165, 1:4,000), p53 (DO-1) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126 HRP; 1:1,000), p73 (Imgenex Corp, San Diego, CA; IMG-259A; 1:1,000) (Bethyl Laboratories, Montgomery, TX; A300-126A; 1:1,000) (Sigma, SAB4300354, 1:1,000), mouse p73 (Santa Cruz, sc-7238, 1:500) (Cell Signaling Technology, Danvers, MA; 4300354; 1:1,000), G6PD (Sigma, HPA000834, 1:3,000), p21 (Cell Signaling, 2947, 1:1,000), and fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (BD Bioscience Pharmingen; 556028; 10 μM).

Techniques: DNA Synthesis, BrdU Incorporation Assay, Transfection, Expressing, Western Blot, Stable Transfection, Plasmid Preparation

Journal: Nature cell biology

Article Title: TAp73 enhances the pentose phosphate pathway and supports cell proliferation

doi: 10.1038/ncb2789

Figure Lengend Snippet: p73 enhances cell growth through up-regulating G6PD ( a ) Proliferation of U2OS cells transfected with control siRNA, p73 siRNA, and G6PD siRNA as indicated. Data are means ± SD (n = 3 independent experiments). Protein expression is shown in Fig. 6 b . ( b ) U2OS cells stably overexpressing G6PD or vector control were transfected with p73 or control siRNA as indicated. Cell proliferation is shown. Data are means ± SD (n = 3 independent experiments). Protein expression in is shown in Supplementary Fig. 4 g . ( c ) Growth of TAp73 +/+ and TAp73 −/− MEF cells stably overexpressing G6PD or vector control. Data are means ± SD (n = 3 independent experiments). Protein expression is shown in Fig. 4 e . ( d ) Growth of TAp73 +/+ and TAp73 −/− MEF cells medium containing 2 mM NAC and four ribonucleosides and four deoxyribonucleosides (Nuc) as indicated (left). Data are means ± SD (n = 3 independent experiments). Representative images of cells stained with crystal violet at day 4 (Right). ( e ) U2OS cells expressing G6PD siRNA or control siRNA were individually injected subcutaneously (SC) in the dorsal flanks of the three nude mice. Tumor weights (mean ± SD, n=3 mice in each group) were measured 3 weeks after inoculation. ( f ) TAp73 +/+ and TAp73 −/− MEF cells (2×10 6 ) stably overexpressing G6PD or vector control were individually injected subcutaneously (SC) in the dorsal flanks of the four nude mice. Tumor weights (mean ± SD, n=4 mice in each group) were measured 3 weeks after inoculation. ( g ) TAp73 +/+ and TAp73 −/− MEF cells (1×10 6 ) expressing G6pd siRNA or control siRNA were individually injected subcutaneously (SC) in the dorsal flanks of the eight nude mice. Half were given water without NAC and the other half were given water with 40 mM NAC throughout the experiment. Tumor weights (mean ± SD, n=8 mice in each group) were measured 3 weeks after inoculation.

Article Snippet: Antibodies against the following proteins/epitopes were used in this study with the company, catalog number, and dilution or concentration indicated: GFP (Clontech Laboratories, Mountain View, CA; 632381; 1:4,000), actin (Sigma-Aldrich, St Louis, MO; A2066; 1:4,000), Flag (Sigma, F3165, 1:4,000), p53 (DO-1) (Santa Cruz Biotechnology, Santa Cruz, CA; sc-126 HRP; 1:1,000), p73 (Imgenex Corp, San Diego, CA; IMG-259A; 1:1,000) (Bethyl Laboratories, Montgomery, TX; A300-126A; 1:1,000) (Sigma, SAB4300354, 1:1,000), mouse p73 (Santa Cruz, sc-7238, 1:500) (Cell Signaling Technology, Danvers, MA; 4300354; 1:1,000), G6PD (Sigma, HPA000834, 1:3,000), p21 (Cell Signaling, 2947, 1:1,000), and fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody (BD Bioscience Pharmingen; 556028; 10 μM).

Techniques: Transfection, Expressing, Stable Transfection, Plasmid Preparation, Staining, Injection

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Eighty-six genes (brown) were down regulated in the absence of all three p53 family members. A number of genes were down regulated in the absence of each p53 family member individually; 109 in p53 deficient cells (red), 148 in p63 deficient cells (yellow), and 131 in p73 deficient cells (blue). Several genes were down regulated in the absence of two family members; 47 in the absence of p53 and p63 (orange), 58 in the absence of p63 and p73 (green), and 41 in the absence of p53 and p73 (purple).

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Each row represents the specified gene. Each column represents the expression level of a specified knock out MEF line relative to the expression level of wild-type MEFs after DNA damage. The red color indicates upregulation, the green color indicates down regulation, while black indicates no significant change of the indicated gene expression. Clustering based on Euclidean distance indicates that p63- and p73- deficient E1A MEFs are more similar to each other than to p53−/− E1A MEFs. Genes of interest are listed in boxes and are associated with their corresponding location on the heatmap.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Expressing, Knock-Out, Gene Expression

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: p53 family response elements assayed by ChIP.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Real time PCR analysis of E1A MEFs of the following genotypes (wild-type, p53−/− , p63−/−, p73−/− and p63−/−;p73−/− ) after treatment with (A) doxorubicin (0.34 µM) for 12 hours or (B) γ radiation (12 hours). The Y-axis shows the fold induction. Bars represent 3 MEF lines for each genotype, each performed in triplicate. Data represent the mean ± SEM. The asterisk denotes statistical significance compared to wild-type, p<0.001.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: (A) Western blot analysis for Rad51 using whole cell lysates from wild-type and p63−/−;p73−/− MEFs treated with 0 Gy or 10 min (m), 30 m, 1 hour (h), 2 h and 4 h after 5 Gy of gamma irradiation. Actin was used as a control for equal loading. (B–I) Immunohistochemistry (IHC) of normal mammary tissue or mammary adenocarcinomas from p63+/−;p73+/− mice using antibodies as follows: (B) normal mammary tissue from p63+/−;p73+/− mouse using Rad51 antibody, (C) mammary adenocarcinoma from p63+/−;p73+/− mouse using Rad51 antibody, (D) normal mammary tissue from p63+/−;p73+/− mouse using BRCA2 antibody, (E) mammary adenocarcinoma from p63+/−;p73+/− mouse using BRCA2 antibody, (F) normal mammary tissue from p63+/−;p73+/− mouse using p63 antibody, (G) mammary adenocarcinoma from p63+/−;p73+/− mouse using p63 antibody, (H) normal mammary tissue from p63+/−;p73+/− mouse using p73 antibody, (I) mammary adenocarcinoma from p63+/−;p73+/− mouse using p73 antibody.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Western Blot, Irradiation, Control, Immunohistochemistry

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) analysis using wild-type E1A MEFs (WT) and E1A MEFs deficient for the p53 family members ( p53−/− , p63−/− and p73−/− ) before (U) and after treatment with doxorubicin (D) for 12 hours. Antibodies used to immunoprecipitate protein-DNA complexes in each cell line are shown in various colors: p53 (red), p63 (blue), and p73 (green). Total input chromatin is shown for each sample (input). Each ChIP was performed using 3 independent MEF lines in triplicate.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Chromatin Immunoprecipitation

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Bar graphs showing fold induction for each luciferase reporter gene in (A–D) p63−/−; p73−/− or (E,F) p53−/−;p73−/− primary MEFs. Reporter genes used are as follows: (A,E) pGL3-Rad51-1 containing the binding elements in intron 1, (B) pGL3-Rad51-2 containing the binding elements in intron 2, (C,F) pGL3-BRCA2 containing the binding element in intron 2, and (D) pGL3-mre-11 containing the binding element in intron 1. Pluses above each bar graph indicate which isoforms of p63 or p73 were transfected in cells with the firefly-luciferase reporter genes. Renilla-luciferase was used as a control for transfection efficiency, and pPERP-luc was used as a positive control. Each experiment was performed 6 times using 3 independent MEF lines. Data are represented as the mean ± SEM.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Luciferase, Binding Assay, Transfection, Control, Positive Control

Journal: Cell death & disease

Article Title: The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells.

doi: 10.1038/cddis.2013.302

Figure Lengend Snippet: Figure 2 p53-family members activate transcription of NIS promoter in liver cancer cell lines. (a) The indicated HCC (HepG2, HUH7 and Hep3B) and CCA (CCSW1 and CCLP1) cell lines were co-transfected with 300 ng of the 2000/ þ 375 NIS promoter luciferase construct and either p53, p63a or p73a expression vectors. Luciferase activity was assayed 24 h after transfection and expressed as fold induction relative to the control after normalization for transfection efficiency using the dual-luciferase assay system. Histograms show the means of at least three experiments performed in quadruplicate. Bars indicate S.D. (b) NIS mRNAs levels were determined by real-time qPCR using NIS-specific primers in HepG2 and CCSW1 cells transfected with the 2000/ þ 375 NIS promoter luciferase construct and the indicated expression vectors. Results are expressed as fold induction relative to the mock-transfected controls after normalization towards endogenous human b-actin mRNAs (beta actin FAM Probe Roche) mRNAs. Data are means±S.D. from at least three independent experiments performed in duplicate. (c) Anti-NIS immunoblot of HepG2 cells transfected with p53, p63a or p73a expression vectors (upper panel). a-Actin protein levels were used to normalize equal loadings from lysate samples. Histograms (lower panel) represent the means of three independent experiments. Bar, S.D. (d) HepG2 cells were co-transfected with the 2000/ þ 375 NIS luciferase construct and 100 nM of p53, p63 and p73 small interfering RNAs or a control siRNA smart pool. Luciferase activity was determined as in (a). Data are means±S.D. of at three independent experiments performed in duplicate. P-values were determined using the two-tails Student’s t-test. *Po0.05; **Po0.01; ***Po0.001

Article Snippet: The following antibodies were used: anti-p53 (DO1) mouse monoclonal, anti-p63 (4A4) rabbit polyclonal from Santa Cruz Biotechnology Inc. (San Diego, CA, USA); anti-p73 mouse monoclonal (IMG-259A) from Imgenex (San Diego, CA, USA), anti-cleaved PARP rabbit polyclonal from Cell Signaling Technology (Danvers, MA, USA); Cell Death and Disease anti-NIS LP10 rabbit polyclonal.9,58 pcDNA-HA-P53, pcDNA-HA-P63a, pcDNAHA-P73a expression vectors are transfected into cells using TransIT-TKO and TransIT-LT1 reagents from Mirus (Mirus Bio LLC).59 pcDNA-NIS expression vectors are described elsewhere.18 The NIS promoter luciferase reporter construct p2.0-NIS-luc was described by Ryu.60 Double-stranded Smart Pool siRNA specific for human NIS, p53, TAp63, TAp73 and control siRNA were purchased from Dharmacon Research (Lafayette, CO, USA) and transfected into cells using TransITTKO and TransIT-LT1 reagents from Mirus (Mirus Bio LLC).

Techniques: Transfection, Luciferase, Construct, Expressing, Activity Assay, Control, Western Blot

Journal: PLoS ONE

Article Title: ΔNp73 Enhances Promoter Activity of TGF-β Induced Genes

doi: 10.1371/journal.pone.0050815

Figure Lengend Snippet: A) Activation of the SBE-luc promoter by a Smad2+Smad4 combination (pink edged bars) and/or ΔNp73 (Black bars) and/or TGF-β1 (right panel). c = empty vector control. B) Activation of the SBE-luc promoter by a Smad3+Smad4 combination (red edged bars) and/or ΔNp73 (black bars) and/or TGF-β1 (right panel). C) Luciferase assay of cells transfected with SBE-luc reporter and ΔNp73 in combination with increasing amounts of (inhibitory) Smad7. Cells in the right panel were also treated with 1 ng/ml TGF-β1.

Article Snippet: Membranes were reacted with the following primary antibodies: α-V5 (Invitrogen) for V5-tagged Smad3, α-HA (Covance) for HA-tagged p73, PAN-p73 IMG-259A (Imgenex), α-GFP (Santa Cruz) for tagged Smad4, and α-Smad3 clone 2C12 (Sigma-Aldrich) for endogenous Smad3, followed by the appropriate HRP-conjugated secondary antibody and ECL detection.

Techniques: Activation Assay, Plasmid Preparation, Luciferase, Transfection

Journal: PLoS ONE

Article Title: ΔNp73 Enhances Promoter Activity of TGF-β Induced Genes

doi: 10.1371/journal.pone.0050815

Figure Lengend Snippet: A) DNA Affinity Precipitation assay (DNAP) of SBE DNA duplexes with extracts of cells left untreated or treated with TGF-β1 after triple transfection with YFP-tagged Smad4, V5-tagged Smad3 and either empty vector control (c), HA-tagged ΔNp73 (ΔN) or HA-tagged TAp73α (TA). B) Relative binding of V5Smad3 to SBE oligos, quantification and normalization for input values of the V5Smad3 results from . C) Relative binding of YFPSmad4 binding to SBE oligos, quantification and normalization for input values of the YFPSmad4 results from . D) Pull-down of SBE oligo incubated with a cell lysate containing HA-ΔNp73 (extract 1) or with a cell-lysate containing V5Smad3 and YFPSmad4 (extract 2) or incubated first with extract 1, washed and incubated with extract 2 (1+2). E) Immunoprecipitation (IP) with α-HA of HA-ΔNp73 induced cells (tetracycline) detecting endogenous Smad3.

Article Snippet: Membranes were reacted with the following primary antibodies: α-V5 (Invitrogen) for V5-tagged Smad3, α-HA (Covance) for HA-tagged p73, PAN-p73 IMG-259A (Imgenex), α-GFP (Santa Cruz) for tagged Smad4, and α-Smad3 clone 2C12 (Sigma-Aldrich) for endogenous Smad3, followed by the appropriate HRP-conjugated secondary antibody and ECL detection.

Techniques: Affinity Precipitation, Transfection, Plasmid Preparation, Binding Assay, Incubation, Immunoprecipitation

Journal: PLoS ONE

Article Title: ΔNp73 Enhances Promoter Activity of TGF-β Induced Genes

doi: 10.1371/journal.pone.0050815

Figure Lengend Snippet: A) Activation of the SBE-luc promoter by a Smad2+Smad4 combination (pink edged bars) and/or ΔNp73 (Black bars) and/or TGF-β1 (right panel). c = empty vector control. B) Activation of the SBE-luc promoter by a Smad3+Smad4 combination (red edged bars) and/or ΔNp73 (black bars) and/or TGF-β1 (right panel). C) Luciferase assay of cells transfected with SBE-luc reporter and ΔNp73 in combination with increasing amounts of (inhibitory) Smad7. Cells in the right panel were also treated with 1 ng/ml TGF-β1.

Article Snippet: Membranes were reacted with the following primary antibodies: α-V5 (Invitrogen) for V5-tagged Smad3, α-HA (Covance) for HA-tagged p73, PAN-p73 IMG-259A (Imgenex), α-GFP (Santa Cruz) for tagged Smad4, and α-Smad3 clone 2C12 (Sigma-Aldrich) for endogenous Smad3, followed by the appropriate HRP-conjugated secondary antibody and ECL detection.

Techniques: Activation Assay, Plasmid Preparation, Control, Luciferase, Transfection

Journal: PLoS ONE

Article Title: ΔNp73 Enhances Promoter Activity of TGF-β Induced Genes

doi: 10.1371/journal.pone.0050815

Figure Lengend Snippet: A) DNA Affinity Precipitation assay (DNAP) of SBE DNA duplexes with extracts of cells left untreated or treated with TGF-β1 after triple transfection with YFP-tagged Smad4, V5-tagged Smad3 and either empty vector control (c), HA-tagged ΔNp73 (ΔN) or HA-tagged TAp73α (TA). B) Relative binding of V5Smad3 to SBE oligos, quantification and normalization for input values of the V5Smad3 results from . C) Relative binding of YFPSmad4 binding to SBE oligos, quantification and normalization for input values of the YFPSmad4 results from . D) Pull-down of SBE oligo incubated with a cell lysate containing HA-ΔNp73 (extract 1) or with a cell-lysate containing V5Smad3 and YFPSmad4 (extract 2) or incubated first with extract 1, washed and incubated with extract 2 (1+2). E) Immunoprecipitation (IP) with α-HA of HA-ΔNp73 induced cells (tetracycline) detecting endogenous Smad3.

Article Snippet: Membranes were reacted with the following primary antibodies: α-V5 (Invitrogen) for V5-tagged Smad3, α-HA (Covance) for HA-tagged p73, PAN-p73 IMG-259A (Imgenex), α-GFP (Santa Cruz) for tagged Smad4, and α-Smad3 clone 2C12 (Sigma-Aldrich) for endogenous Smad3, followed by the appropriate HRP-conjugated secondary antibody and ECL detection.

Techniques: Affinity Precipitation, Transfection, Plasmid Preparation, Control, Binding Assay, Incubation, Immunoprecipitation

Journal: eLife

Article Title: Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase Trim63

doi: 10.7554/eLife.10528

Figure Lengend Snippet: ( A ) Proteins from muscles were immuno-precipitated with a p63 antibody and then separated on a 10% SDS PAGE gel. Western blot experiment was performed using an antibody against p63 total. Shows pools of proteins from 3 animals at 105d. TBP was used as loading control. ( B ) Proteins (40 µg) from muscles were separated on 10% SDS PAGE gel. Western blot probing was performed with p53 antibody (IC12, 1/2000, Cell Signaling, Danvers, MA) and True Blot (Rockland Immunochemicals, Pottstown, PA) secondary antibody avoiding Ig heavy chain recognition. Tubilin was used as loading control. Graph below shows% of induction relative to the mean of p53 expression level in WT animals normalised with tubulin. DOI: http://dx.doi.org/10.7554/eLife.10528.011

Article Snippet: *p<0.01. ( C, D ) Chromatin immunoprecipitation (ChIP) assay was performed on the Trim63 promoter using RT-qPCR on RE1/2 and RE4. p53 immunoprecipitation (C) was performed using p53 antibody IC12 (Cell Signalling), p73 immunoprecipitation was performed using p73 antibody IMG-259a (Imgenex).

Techniques: SDS Page, Western Blot, Expressing

Journal: eLife

Article Title: Transcriptional activator TAp63 is upregulated in muscular atrophy during ALS and induces the pro-atrophic ubiquitin ligase Trim63

doi: 10.7554/eLife.10528

Figure Lengend Snippet: ( A ) mRNA levels of Trim63 in C2C12 cells following transfection with siRNA control and siRNA directed against p73, p53 and a mix of siRNA against P53, and the TA isoforms of Trp63 and P73 (siMIX). Bars represent means (relative induction versus Ct) with standard deviation (n = 3). *p<0.01. ( B ) mRNA level for TA isoforms of Trp63 , TA isoforms of P73 and P53 in C2C12 cells following transfection with siRNA control and siRNA directed against p63, p73, and p53. Bars represent means (relative induction versus Ct) with standard deviation (n = 3). *p<0.01. ( C, D ) Chromatin immunoprecipitation (ChIP) assay was performed on the Trim63 promoter using RT-qPCR on RE1/2 and RE4. p53 immunoprecipitation (C) was performed using p53 antibody IC12 (Cell Signalling), p73 immunoprecipitation was performed using p73 antibody IMG-259a (Imgenex). Bars correspond to means with SD (n = 3). *p<0.01 as calculated by a one-way ANOVA test followed by a Tukey post-test. DOI: http://dx.doi.org/10.7554/eLife.10528.019

Article Snippet: *p<0.01. ( C, D ) Chromatin immunoprecipitation (ChIP) assay was performed on the Trim63 promoter using RT-qPCR on RE1/2 and RE4. p53 immunoprecipitation (C) was performed using p53 antibody IC12 (Cell Signalling), p73 immunoprecipitation was performed using p73 antibody IMG-259a (Imgenex).

Techniques: Transfection, Standard Deviation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Immunoprecipitation