anti polyclonal rabbit 153 against phospho-p53 (ser15)  (Cell Signaling Technology Inc)

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Targeting strategy. The wildtype (WT) Trp53 gene is within a 17-kb-long EcoRI (RI) fragment (black boxes are for coding sequences and white boxes for UTRs). The targeting construct contains: (1) a 1.5-kb-long 5’ homology region; (2) a Lox-Stop-Lox (LSL) cassette with a neomycin selection gene (Neo), four transcriptional stops (STOP) and an EcoRI site, flanked by LoxP sites (arrowheads); (3) p53 coding exons, including the Y217C (YC) missense mutation in exon 6 (asterisk) and an additional BanII site; (4) a 2.8-kb-long 3’ homology region; and (5) the diphteria α-toxin (DTA) gene for targeting enrichment. Proper recombinants with a Trp53 LSL-Y217C allele, resulting from the described crossing-overs, were G418 resistant. They were identified by a 2.4-kb-long band after PCR with primers a and b, and confirmed by bands of 635 and 224 bp after PCR with primers c and d and BanII digestion. They were also verified by Southern blot with the indicated probe as containing a 10.5 kb EcoRI band. Two recombinant ES clones were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping with primers c and d and BanII digestion. Excision of the LSL cassette was performed in vivo, by breeding Trp53 +/LSL-Y217C male mice with females carrying the PGK- Cre transgene, to obtain mice with a Trp53 Y217C allele. ( B–D ) Screening of recombinant ES clones (+) by PCR with primers a and b ( B ); PCR with primers c and d then BanII digestion ( C ); Southern blot ( D ). ( E ) Genotyping of mouse embryonic fibroblasts (MEFs) from an intercross of Trp53 +/Y217C mice, by PCR with primers c and d and BanII digestion. ( F ) Trp53 Y217C sequence around codon 217. The introduced Y217C missense mutation and the silent mutation creating an additional BanII restriction site are highlighted (asterisks). ( G ) WT and Trp53 Y217C/Y217C (YC/YC) MEFs express similar p53 mRNA levels. Total RNA was extracted, then p53 mRNAs were quantified by real-time qPCR, normalized to control mRNAs and the amount in WT cells was assigned the value of 1. Means + SEM (n=3) are shown. Primer sequences are listed in . Figure 1—source data 1. Labeled files for gels and blots in . Figure 1—source data 2. Raw and unedited gels and blots for .

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Construct, Selection, Mutagenesis, Southern Blot, Recombinant, Clone Assay, Injection, Transmission Assay, In Vivo, Sequencing, Control, Labeling

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: Portions of the DNA-binding domains from the mouse (residues 208–228) and human (residues 211–231) p53 proteins are shown, with identical residues in bold, and mouse Tyrosine 217 and human Tyrosine 220 in red.

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Binding Assay

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Increased p53 protein levels in Trp53 YC/YC and Trp53 +/YC mouse embryonic fibroblasts (MEFs). MEFs of the indicated genotypes were treated or not with 10 μM Nutlin 3a for 24 hr, then protein extracts were immunoblotted with antibodies against Mdm2, p53, p21, and actin. ( B ) The transactivation of classical p53 target genes Cdkn1a and Mdm2 is impaired in Trp53 YC/YC cells. Wildtype (WT), Trp53 YC/YC , and Trp53 -/- MEFs were treated as in ( A ), then (top) mRNAs were quantified in five to six independent experiments using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in unstressed WT cells assigned a value of 1; or (bottom) ChIP assays were performed at the Cdkn1a and Mdm2 promoters in two to three independent experiments with an antibody against p53 or rabbit IgG as a negative control. Immunoprecipitates were quantified using real-time PCR, normalized to data over an irrelevant region, and the amount in unstressed WT cells was assigned a value of 1. Error bars: SEM. ( C ) Assessment of p53 WT and p53 Y217C subcellular localization by cellular fractionation. WT and Trp53 YC/YC MEFs were treated or not with 1 μΜ doxorubicin (Doxo) for 24 hr, submitted to cellular fractionation, then protein extracts were immunoblotted with antibodies against p53 or the fraction controls Tubulin for cytoplasm (Cp.), Nup98 for nucleoplasm (Np.), and histone H3 for chromatin (χin). ( D ) Assessment of p53 WT and p53 Y217C subcellular localization by immunofluorescence. WT, Trp53 YC/YC and Trp53 -/- MEFs were treated with 10 μM Nutlin 3a for 24 hr, then stained with antibodies against p53 (red) or actin (green) and DNA was counterstained with DAPI (blue). ( E ) Absence of a cell cycle arrest response in Trp53 YC/YC MEFs. Asynchronous cell populations of Trp53 +/+ , Trp53 YC/YC , and Trp53 -/- MEFs were analyzed 24 hr after 0, 3, or 12 Gy γ-irradiation. Means + SEM from three independent experiments. ( F ) Absence of a p53-dependent apoptotic response in Trp53 YC/YC thymocytes. Age-matched mice of the indicated genotypes were left untreated or submitted to 10 Gy whole-body γ-irradiation then sacrificed after 4 hr and their thymocytes were stained with Annexin V-FITC and analyzed by FACS. Means + SEM from two independent experiments. ( G ) Increased chromosomal instability in Trp53 YC/YC fibroblasts. Metaphase spreads were prepared from WT, Trp53 YC/YC , and Trp53 -/- MEFs at passage 4, then aberrant metaphases (with chromosome breaks, radial chromosomes, or double-minute chromosome [DMs]) were scored. Left: distribution of aberrant metaphases. Data from 110 WT, 97 Trp53 YC/YC , or 119 Trp53 -/- complete diploid metaphases, independently observed by two experimenters. Right: examples of two aberrant Trp53 YC/YC metaphases: one with a DM, a chromosome break (Br) and a radial chromosome (R), the other with multiple DMs. Enlargements of regions of interest are presented between the two metaphases. Scale bars ( D, G ): 5 μm. ***p<0.001, **p<0.01, *p<0.05, °p=0.09, ns: non-significant by Student’s t ( B, E, F ) or Fisher’s ( G ) tests. Figure 2—source data 1. Labeled files for gels and blots in . Figure 2—source data 2. Raw and unedited gels and blots for .

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Real-time Polymerase Chain Reaction, Control, Negative Control, Cell Fractionation, Immunofluorescence, Staining, Irradiation, Labeling

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Transactivation of the classical p53 target genes Cdkn1a (alias p21 ), Mdm2, and Pmaip1 (alias Noxa ) in response to Nutlin or Doxorubicin. WT, Trp53 +/YC , and Trp53 +/- mouse embryonic fibroblasts (MEFs) were treated or not with 10 μM Nutlin 3a (Nut) or 1 μM Doxorubicin (Doxo) for 24 hr, then mRNAs were quantified in ≥4 independent experiments using real-time PCR, with results normalized to control mRNAs and mean RNA amounts in unstressed WT cells assigned a value of 1. Means + SEM are shown. For each condition and gene, a dominant-negative effect (DNE) would lead to significant decrease in transactivation in Trp53 +/YC MEFs compared to both WT and Trp53 +/- MEFs, a result that was not observed. ( B ) Cell cycle arrest responses to γ-irradiation. Asynchronous cell populations of WT, Trp53 +/YC , and Trp53 +/- MEFs were analyzed 24 hr after 0, 3, or 12 Gy γ-irradiation. Means + SEM from three independent experiments. The comparison of cells submitted to identical irradiation doses revealed similar arrest responses in cells of all genotypes. ( C ) Apoptotic responses to γ-irradiation. WT, Trp53 +/YC , and Trp53 +/- MEFs age-matched mice were left untreated or submitted to 10 Gy whole-body γ-irradiation, then sacrificed after 4 hr and their thymocytes were stained with Annexin V-FITC and analyzed by FACS. Means + SEM from two independent experiments. The percentage of apoptotic cells was significantly higher in irradiated WT thymocytes compared to irradiated Trp53 +/YC or Trp53 +/- cells, whereas Trp53 +/YC and Trp53 +/- cells were not significantly different. **p<0.01, *p<0.05, ns: non-significant by Student’s t-test.

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Real-time Polymerase Chain Reaction, Control, Dominant Negative Mutation, Irradiation, Comparison, Staining

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Distribution of weaned mice obtained from Trp53 +/- or Trp53 +/YC intercrosses. Obs: observed numbers of mice at weaning (P21); exp: expected numbers assuming a Mendelian distribution without sex distortion; f/m: observed female/male ratios. Consistent with previous reports, the observed distribution of weaned mice from Trp53 +/- intercrosses did not conform to values expected for a Mendelian distribution without sex distortion (U=5; χ 2 =16.31>15.09), indicating a significant deficit in female Trp53 -/- mice (top). The distribution of weaned mice from Trp53 +/YC intercrosses diverged even more from values for a Mendelian distribution without sex distortion (U=5; χ 2 =104.23>15.09), due to a striking deficit in female Trp53 YC/YC mice (bottom). Differences between the frequencies of Trp53 YC/YC (4/677) and Trp53 -/- (8/196) females in the progeny, or between the female to male ratios for Trp53 YC/YC (4/75) and Trp53 -/- (8/28) animals, are statistically significant (p=0.0012 and p=0.0087 in Fisher’s tests, respectively). ( B ) Exencephaly is frequently observed in p53 YC/YC female embryos. Top: the distribution of E12.5–16.5 embryos from heterozygous ( Trp53 +/YC ) intercrosses or heterozygous-homozygous ( Trp53 +/YC × Trp53 YC/YC ) crosses is shown. f or m exenc.: number of female or male embryos with exencephaly; o.a.: embryos with other abnormalities. Below, examples of female Trp53 YC/YC embryos at E12.5, E13.5, and E16.5 exhibiting exencephaly (arrows) are each shown (center) together with a normal embryo from the same litter (bottom). Scale bars : 1 mm. ( C ) Distribution of mice at birth from the indicated crosses. Of note, out of five Trp53 YC/YC females observed at birth, only one remained alive at weaning age. Thus, the female/male ratio for weaned Trp53 YC/YC animals from these crosses was 1/22, a ratio similar to the one observed in A (4/75).

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques:

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A–B ) In homozygous males, p53 Y217C leads to accelerated tumor-induced death ( A ), and aggressive metastatic tumors ( B ); n=cohort size. ( C ) Hematoxylin and eosin (H&E) staining of sections from the lung (top) and spleen (bottom) of Trp53 -/- and Trp53 YC/YC male mice, showing metastases in Trp53 YC/YC animals. Normal organ structures are shown, with ‘A’ indicating pulmonary alveoli, and ‘WP’ and ‘RP’ standing for splenic white and red pulp, respectively. In the lung section of the Trp53 YC/YC mouse, the rectangle indicates a lymphoma area. In the spleen section of the Trp53 YC/YC mouse, the typical splenic structures are absent due to massive tissue homogenization of the spleen by lymphoma cells.

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Staining, Tissue Homogenization

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: ( A ) Heat-map plot, with 717 differentially expressed genes suggestive of a loss of function (LOF), a separation of function (SOF), or a gain of function (GOF) for the p53 Y217C mutant, ranked according to log 2 fold change (n=number of genes). ( B ) Evidence of LOF in Trp53 YC/YC cells for genes encoding Puma, p21, and Zmat3. Data from three mice per genotype. Means + SEM are shown. ***p<0.001, **p<0.01, *p<0.05, °p=0.07, ns: non-significant by Student’s t-tests.

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Mutagenesis

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet: Effects of the Trp53 Y217C mutation: a summary. The comparison between Trp53 -/- and Trp53 Y217C/Y217C mice is presented. The phenotypes observed in Trp53 -/- male (M) and female (F) mice result from p53 loss of function (LOF), whereas those observed in Trp53 Y217C/Y217C mice result from p53 LOF as well as additional effects (gain of function [GOF] in bold). The + signs denote the presence of a phenotype. Xi: X chromosome inactivation.

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Mutagenesis, Comparison

Journal: eLife

Article Title: Oncogenic and teratogenic effects of Trp53 Y217C , an inflammation-prone mouse model of the human hotspot mutant TP53 Y220C

doi: 10.7554/eLife.102434

Figure Lengend Snippet:

Article Snippet: ChIP analysis was performed as previously described ( ). p53-DNA complexes were immunoprecipitated from total extracts by using 50 μg of a polyclonal antibody against p53 (FL-393, Santa Cruz Biotechnology) and 300 μg of sonicated chromatin.

Techniques: Cell Culture, Sequencing, Recombinant, SYBR Green Assay, Staining, Software