Journal: Cancer Research

Article Title: The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

doi: 10.1158/0008-5472.can-20-1750

Figure Lengend Snippet: Figure 1. Generation of germline p53-R334H mouse model. A, Targeting strategy. The p53R334H point mutation in exon 10 is marked by an asterisk. B, Identification of targeted ES cell clones by PCR amplification using an outside probe yielding a 3.6 kb WT band and a 4.9 kb mutant band. C, Genotyping of animals using allele-specific primers results in a 109 bp WT band and a 128 bp mutant p53 band. PCR products were resolved on a 3% LMP agarose gel. D, Sequencing of the p53 missense mutation from genomic DNA. E, Genotype analysis of offspring from p53R334H heterozygous crosses.

Article Snippet: Cell lysates were prepared, and total cellular protein was subjected to immunoprecipitation using rabbit polyclonal anti-p53 antibody (FL393; Santa Cruz Biotechnology) followed by immunoblotting as described above using sheep polyclonal anti-p53 (AP05006PU-N; Acris Antibodies, Inc.), mouse monoclonal anti-Mdm2 (2A10, cat. #ab16895; Abcam), and mouse monoclonal anti–b-actin (cat. #A5441; Sigma).

Techniques: Mutagenesis, Clone Assay, Agarose Gel Electrophoresis, Sequencing

Journal: Cancer Research

Article Title: The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

doi: 10.1158/0008-5472.can-20-1750

Figure Lengend Snippet: Figure 2. Mutant p53R334H/R334H mice develop tumors with long latency and incom- plete penetrance. A, Kaplan–Meier tumor-free survival curves. B, Histopa- thology of tumors arising in homozy- gous p53R334H mouse (20 months of age). Osteosarcoma, vertebra, hema- toxylin and eosin (2.5, top left plot), osteosarcoma, vertebra, hematoxylin and eosin (40, top middle plot), oste- osarcoma, p53 IHC (40, top right plot); mammary adenocarcinoma, hematoxy- lin and eosin (40, bottom left plot) mammary adenocarcinoma, p53 IHC (40, bottom right plot). C, Histopa- thology of primary and metastatic sar- coma arising in homozygous p53R334H mouse (21 months of age). Angiosar- coma with inset highlighting microcapil- lary formation with red blood cells (40, left), angiosarcoma CD31 staining (40, middle), and metastatic angiosar- coma attached to blood vessel wall (10, right).

Article Snippet: Cell lysates were prepared, and total cellular protein was subjected to immunoprecipitation using rabbit polyclonal anti-p53 antibody (FL393; Santa Cruz Biotechnology) followed by immunoblotting as described above using sheep polyclonal anti-p53 (AP05006PU-N; Acris Antibodies, Inc.), mouse monoclonal anti-Mdm2 (2A10, cat. #ab16895; Abcam), and mouse monoclonal anti–b-actin (cat. #A5441; Sigma).

Techniques: Mutagenesis, Staining

Journal: Cancer Research

Article Title: The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

doi: 10.1158/0008-5472.can-20-1750

Figure Lengend Snippet: Figure 3. Mutant p53-R334H is abnormally expressed and functionally impaired in thymocytes. A, FACS analysis of isolated thymocytes in cell culture either untreated (Control) or 16 hours post IR (5 Gy g-IR). Results are representative of four independent experiments. B, Western blot analysis of isolated thymocytes in cell culture at 0 (untreated), 2, and 5 hours post g-IR (5 Gy). Proteins were detected using the following primary antibodies: p53 (1C12); p21 (F5); Puma (P4743); b-actin (AC-15). C, Western blot analysis of tissues from WT, p53R334H/R334H, p53R172H/R172H, andp53/ mice either untreated (Control) or 6 hours after treatment with 5 Gy whole-body IR. Each sample represents an individual animal. Proteins were detected using the following primary antibodies: p53 (1C12); p21 (F5); b-actin (AC-15). D, IHC analysis of p53 in thymus from mice either untreated or 6 hours after treatment with 5 Gy whole-body IR. p53 was detected using rabbit polyclonal CM5 antibody (Leica).

Article Snippet: Cell lysates were prepared, and total cellular protein was subjected to immunoprecipitation using rabbit polyclonal anti-p53 antibody (FL393; Santa Cruz Biotechnology) followed by immunoblotting as described above using sheep polyclonal anti-p53 (AP05006PU-N; Acris Antibodies, Inc.), mouse monoclonal anti-Mdm2 (2A10, cat. #ab16895; Abcam), and mouse monoclonal anti–b-actin (cat. #A5441; Sigma).

Techniques: Mutagenesis, Isolation, Cell Culture, Control, Western Blot

Journal: Cancer Research

Article Title: The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

doi: 10.1158/0008-5472.can-20-1750

Figure Lengend Snippet: Figure 4. Mutant p53-R334H transcriptional response is attenuated in thymocytes in vivo in response to DNA damage. Affymetrix gene expression analysis of WT and R334H thymocytes either untreated (Control) or 3 hours after treatment with 5 Gy whole-body IR. Each lane represents an individual mouse. A, Heat map depicting a global perspective of all gene profiles measured by the microarray. B, Heat map display of 48 core enrichment genes in the Hallmark p53 pathway that are activated in WT cells following radiation (GSEA enrichment score 2.66, P < 0.001, FDR < 0.001). C, Heat map display of 41 core enrichment genes in the Hallmark MYC targets V2 that are activated in WT cells following radiation (FDR < 0.001). D, Quantitative real-time analysis of WT and p53-R334H thymocytes either untreated (Control) or 3 hours after treatment with 5 Gy whole-body IR (n ¼ 3).

Article Snippet: Cell lysates were prepared, and total cellular protein was subjected to immunoprecipitation using rabbit polyclonal anti-p53 antibody (FL393; Santa Cruz Biotechnology) followed by immunoblotting as described above using sheep polyclonal anti-p53 (AP05006PU-N; Acris Antibodies, Inc.), mouse monoclonal anti-Mdm2 (2A10, cat. #ab16895; Abcam), and mouse monoclonal anti–b-actin (cat. #A5441; Sigma).

Techniques: Mutagenesis, In Vivo, Gene Expression, Control, Microarray

Journal: Cancer Research

Article Title: The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

doi: 10.1158/0008-5472.can-20-1750

Figure Lengend Snippet: Figure 5. Mutant p53-R334H is functional in MEFs in response to DNA damage and in vitro senescence. A, Mutant p53-R334H confers short-term proliferative advantage in low passage MEFs. Curves represent the average of three independent MEF cell lines for each genotype counted daily in duplicate (P ¼ 0.002) B, Mutant p53-R334H competently induced cellular senescence in serially passaged MEFs (P ¼ 0.008). C, Mutant p53-R334H induces target gene expression in response to DNA damage induced by 10 mmol/L etoposide at 4 hours after treatment. Proteinswere detected using the primary antibodies p53 (PAb7); p21 (C19). D, Mutant p53-R334H induces target gene expression in response to DNA damage induced by 30 J UV radiation at 24 hours. Proteins detected using the primary antibodies p53 (1C12); p21 (F-5). MEFs at passage 4 were used in experiments presented in C and D.

Article Snippet: Cell lysates were prepared, and total cellular protein was subjected to immunoprecipitation using rabbit polyclonal anti-p53 antibody (FL393; Santa Cruz Biotechnology) followed by immunoblotting as described above using sheep polyclonal anti-p53 (AP05006PU-N; Acris Antibodies, Inc.), mouse monoclonal anti-Mdm2 (2A10, cat. #ab16895; Abcam), and mouse monoclonal anti–b-actin (cat. #A5441; Sigma).

Techniques: Mutagenesis, Functional Assay, In Vitro, Targeted Gene Expression

Journal: Cancer Research

Article Title: The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

doi: 10.1158/0008-5472.can-20-1750

Figure Lengend Snippet: Figure 6. Mutant p53-R334H has a prolonged half-life and is compromised in forming stable tetramers in primary MEFs. A, Cycloheximide half-life experiment. Early passage MEFs (passage 5) were treated with 10 mg/mL cycloheximide and harvested at indicated time points to determine p53 levels by Western blot analysis using mouse mAb 1C12. B, Protein complex analysis of primary MEFs by size exclusion chromatography and Western blot analysis (see Materials and Methods). P53 protein was detected using mAb 1C12. C, SaOS2 cells transfected with CMV Neo vector without an insert, p53 WT, or p53-R334H cDNA. Coimmunoprecipitation using Mdm2 (2A10) Ab and blotted for p53 using Sheep anti-p53 polyclonal Ab.

Article Snippet: Cell lysates were prepared, and total cellular protein was subjected to immunoprecipitation using rabbit polyclonal anti-p53 antibody (FL393; Santa Cruz Biotechnology) followed by immunoblotting as described above using sheep polyclonal anti-p53 (AP05006PU-N; Acris Antibodies, Inc.), mouse monoclonal anti-Mdm2 (2A10, cat. #ab16895; Abcam), and mouse monoclonal anti–b-actin (cat. #A5441; Sigma).

Techniques: Mutagenesis, Western Blot, Size-exclusion Chromatography, Transfection, Plasmid Preparation

Journal: Cancer Research

Article Title: The Common Germline TP53-R337H Mutation Is Hypomorphic and Confers Incomplete Penetrance and Late Tumor Onset in a Mouse Model

doi: 10.1158/0008-5472.can-20-1750

Figure Lengend Snippet: Figure 7. Mutant p53-R334H functions as a tumor suppressor in Em-Myc lympho- magenesis. A, Kaplan–Meier survival of Em-Myc transgenic WT, p53WT/R334H, and p53R334H/R334H mice. B, Western blot analysis of B-cell lymphomas aris- ing in Em-Myc WT, p53WT/R334H, and p53R334H/R334H mice. C, Characteriza- tion of p53 and p19Arf status in B-cell lymphomas arising in Em-Myc WT, p53WT/R334H, and p53R334H/R334H mice. Proteins were detected by Western blot analysis using the following prima- ry antibodies: p53 (1C12); p19Arf (ab80; ABCAM).

Article Snippet: Cell lysates were prepared, and total cellular protein was subjected to immunoprecipitation using rabbit polyclonal anti-p53 antibody (FL393; Santa Cruz Biotechnology) followed by immunoblotting as described above using sheep polyclonal anti-p53 (AP05006PU-N; Acris Antibodies, Inc.), mouse monoclonal anti-Mdm2 (2A10, cat. #ab16895; Abcam), and mouse monoclonal anti–b-actin (cat. #A5441; Sigma).

Techniques: Mutagenesis, Transgenic Assay, Western Blot

Journal: Biomedicines

Article Title: An Increased Plasma Level of ApoCIII-Rich Electronegative High-Density Lipoprotein May Contribute to Cognitive Impairment in Alzheimer’s Disease

doi: 10.3390/biomedicines8120542

Figure Lengend Snippet: Analysis of function and proinflammatory properties of HDL. ( A ) Function of reverse cholesterol efflux was decreased in HDL from AD patients. ( B ) AD-HDL induced vibrant expression of ganglioside GM1 on the membrane of marine macrophages, RAW 264.7 cells. AD-HDL enhanced the higher expression levels of ( C ) LOX-1 on the membrane and ( D ) TNF-α in the conditioned culture media. * p < 0.05, ** p < 0.01. Abbreviations. AD: Alzheimer’s disease; PBS: phosphate-buffered saline; Ctl-HDL: HDL from controls; AD-HDL: HDL from AD patients; ABCA1: ATP-binding cassette, sub-family A member 1; LOX-1: lectin-type oxidized LDL receptor; TNF-α: tumor necrosis factor-α.

Article Snippet: The primary antibodies were ABCA1 (1:100, GeneTex, California, USA), a lectin-type oxidized LDL receptor (LOX-1) (1:1000, Origene, Rockville, USA), phosphor-NF-κB p65 (1:1000, GeneTex) and β-actin (1:5000, Sigma-Aldrich).

Techniques: Expressing, Membrane, Saline, Binding Assay