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  • 99
    Thermo Fisher p53
    The level of huntingtin protein is lower in mice deficient in <t>p53</t>
    P53, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore anti p53
    (A) The MYCN-ChIP-Seq database and <t>p53-ChIP-Seq</t> composite database were analyzed to determine binding patterns at the MYCN (E-box) and p53 (p53-RE) promoters in the top genes. The p53-repressed and p53-activated genes under low MYCN or high MYCN conditions were analyzed and summarized here for the presence or absence of E-box and p53-REs. Top 30 genes in each category by fold change were analyzed. Red= MYCN E-box present; Blue= p53-RE present. (B) Functional annotations of p53 response genes under high MYCN condition were analyzed and found to be highly correlated with worse prognosis and MYCN levels in NB patients. R2: Kosak (n=498) dataset of NB patient was used.
    Anti P53, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 1407 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology p53
    M induces nuclear localization of a <t>p53</t> NLS mutant (p53 KRKKK ) and this is dependent on the nuclear localization signal of M and efficient interaction between p53 and the M protein. A. p53−/− HCT116 cells were transfected with the plasmids indicated. Localization of full-length p53 was determined by immunofluorescence. B. Quantification of immunofluorescence staining in A. 300 cells per transfection condition were scored as having primarily nuclear p53, primarily cytoplasmic p53, or both nuclear and cytoplasmic p53. Values are the average of three independent experiments and error bars represent standard error. * p
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    99
    Cell Signaling Technology Inc p53
    PON2 reduces the IGF-1 level via C-Jun transcription factor in ID8 cells. ID8 EV and ID8 hPON2 cell culture lysates were prepared and utilized for chromatin immunoprecipitation assays with C-Jun ( a ) and <t>p53</t> ( b ) antibodies. c) Equal number of ID8 hPON2 cells were plated in six-well plates. At 60 % confluence level, ID8 hPON2 cells were transfected with either hPON2 siRNA or scrambled siRNA, and PON2 expression analyzed by western blotting. d) Data in c are quantified using ImageJ software. e) Four six-well plates of cells were pooled from PON2-siRNA-treated ID8 hPON2 or scrambled siRNA-treated ID8 hPON2 cells. Chromatin immunoprecipitation assay was performed and C-Jun promoter was quantified ( e ) as described in the materials and methods section. Cell culture supernatants obtained from cells described under ( e ) and IGF-1 level was analyzed by ELISA ( f ), g ) cell proliferation, and h) mitochondrial superoxide levels were measured as described in the method section. * p
    P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 8090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology anti p53
    Genotypes of Control Embryos in Figures and the Genders Associated with Phenotypes (a) Table identifying the genotypes of control embryos shown for each analysis. (b) The table shows the number of male and female <t>p53</t> 25,26,53,54/+ embryos observed with the indicated phenotypes, as assessed by Zfy PCR. Phenotypes are well represented in both sexes.
    Anti P53, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 6212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies p53
    Ex vivo analysis of tumour slices by two-photon microscopy. ( a ) Ex vivo tumour slice from <t>NICD/p53</t> −/− mice observed by two-photon microscopy and represented as mosaic of 9 × 9 connected tiles. Epithelial cells have GFP-positive nuclei (green), second harmonic generation (SHG, pink) reveals the presence of collagen I. Insets represent higher magnification of several tumour areas (1–3 tumour bulk; 4–6 invasive areas). Scale bar, 100 μm. ( b – d ) Several examples of ex vivo tumour slices from NICD/p53 −/− mice. Epithelial cells (GFP-positive nuclei, green), collagen I (SHG, pink), all cells (membrane dye, FM 4-64, blue). Images are taken from a Z-stack with 6 μm between planes. ( b ) Invasive front of the tumour. Note that stromal cells surround cancer cells. ( c ) Cancer cells invading stroma as clusters and strings parallel to the collagen fibres. ( d ) Cluster and single cancer cells invading collagen-rich stroma. Single cancer cells are labelled with white arrowheads. Inset, higher magnification of individual cancer cell with elongated nucleus. Scale bar, 100 μm.
    P53, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 3301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti p53
    6-Sho enhances TRAIL-mediated <t>p53</t> expression. Huh7 cells were treated with 6-sho (5, 10 and 20 µ M) for 18 h. (A) p53 levels assessed by western blot analysis using β-actin as control. (B) Cells stained with p53 antibody (red) and DAPI nuclear stain (blue) evaluated using fluorescence microscopy (magnification, ×400). Western blot analysis of p53 levels in Huh7 cells were pretreated with (C) CQ (10 mM) for 1 h or (D) NAC (10 mM) for 1 h followed by CQ (10 mM) for 1 h prior to exposure to 6-sho (20 µ M) for 18 h and TRAIL (200 ng/ml) for 2 h. 6-Sho, 6-shogaol; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; CQ, chloroquine; p53, tumor-suppressor protein 53; NAC, N -acetyl-L-cysteine.
    Anti P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2784 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p53  (Abcam)
    99
    Abcam p53
    Effects of TUN treatment on glioma cell apoptosis. Cells were treated with TUN (2 mg/ml) or vehicle control. (A and B) Apoptosis rate following treatments for (A) BV-2 (A) and (B) BC3H1 cells. (C and D) Western blotting results for expression of cleaved PARP and caspase-9 in (C) BV-2 and (D) BC3H1 (D) cells. (E and F) Western blotting results for expression of Bcl-2 and <t>P53</t> in (E) BV-2 and (F) BC3H1 cells. **P
    P53, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 2561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc phospho p53
    Apigenin administration inhibits 22Rv1 tumor growth in athymic nude mice via modulation of <t>p53</t> signaling pathways. Approximately 1 million cells were injected into both flanks of each mouse to initiate ectopic prostate tumor growth, and apigenin was provided to the animals 2 weeks before cell inoculation. Mice were fed ad libitum with Teklad 8760 autoclaved high-protein diet. Apigenin was provided with 0.5% methyl cellulose and 0.025% Tween 20 as vehicle to these animals perorally on a daily basis. Group I, control, received 0.2 ml vehicle only, Group II received 20 μg apigenin per mouse in 0.2 ml vehicle, and Group III received 50 μg apigenin per mouse in 0.2 ml vehicle daily for 8 weeks. Once the tumor xenografts started growing, their sizes were measured twice weekly in two dimensions throughout the study. (A) Tumor volume (mm 3 ) in control and treated groups. (B) Wet weight of tumors is represented as the mean of 8–10 tumors from each group. (C) Apoptosis as demonstrated by ELISA for 22Rv1 tumors after apigenin intake at the indicated doses. Values are means±SE, n =6–8, repeated twice with similar results. (D) Immunoblots for p53, Ser15-p53, cytochrome c, Bax, Bcl-2, and caspase-3 in tumor lysates after apigenin intake at the indicated doses. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. Significantly different from control: ** p
    Phospho P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti tp53
    Venn diagram depicting overlap between up-regulated and down-regulated genes when comparing homozygous mutant tp53M214K/M214K and <t>tp53</t> del/del MPNST to whole adult zebrafish.
    Anti Tp53, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novocastra p53
    <t>p53</t> restoration induces senescence in premalignant cyclin D1-expressing pineal cells. (A and B) Shown are representative staining for BrdU along with the corresponding DAPI nuclear stain (A) and senescence-associated beta-galactosidase (SABG) staining (B) in Irbp-CyclinD1 , p53ERTAM Ki /− pineal cells explanted at P10 and treated for 7 days with either vehicle (Vh) or 4OHT to restore p53, as indicated. The bottom panels show percent BrdU-positive cells (A) and SABG-positive area (measured in pixel density, as explained in Materials and Methods) (B) under each condition, as indicated. Each point represents the means from 3 independent experiments. (C) Representative immunostaining for the indicated proteins in pineal gland sections from Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− mice treated for 10 days (P60 to P70) with Vh or tamoxifen (TAM) and, as controls, Irbp-Cyclin D1 , p53 −/− mice treated with TAM (p53−/− + TAM). (D) Number of Ki67-positive cells per field under each condition shown in panel C as indicated. Each point represents the means using at least 4 pineal glands. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P
    P53, supplied by Novocastra, used in various techniques. Bioz Stars score: 92/100, based on 1125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti phospho p53
    WRN depletion activates a <t>p53</t> response in MSI cells. a, Phospho-p53 (S15) IF following sgRNA transduction in ovarian cell lines (50 µm scale bar). b, Nuclear phospho-p53 (S15) staining intensity per cell following WRN knockout compared to control sgRNA. Mean log fold-change: 0.059 (OVK18), −0.037 (ES2). Difference in log fold-change between OVK18 and ES2; P value (contrast test of least-squares means)
    Anti Phospho P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 577 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho p53 ser15
    WRN depletion activates a <t>p53</t> response in MSI cells. a, Phospho-p53 (S15) IF following sgRNA transduction in ovarian cell lines (50 µm scale bar). b, Nuclear phospho-p53 (S15) staining intensity per cell following WRN knockout compared to control sgRNA. Mean log fold-change: 0.059 (OVK18), −0.037 (ES2). Difference in log fold-change between OVK18 and ES2; P value (contrast test of least-squares means)
    Phospho P53 Ser15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 745 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti p53 antibodies
    Complex reduces proliferation of MCF7 cell line and enhances expression of <t>p53</t> target genes in p53-dependent manner. a Complex negatively affects viability of MCF7 p53wt and MCF7 p53 –/– tumor cells. MCF7 p53wt (dark gray), MCF7 p53 −/− (light gray) cells were treated with Complex (50 μM), Ligand (50 μM and 100 μM), DMSO (1%, vehicle control), and doxorubicin (4 μM) for 24 h. Cell viability was evaluated by colorimetric MTS assay. Ligand demonstrated no cytotoxicity for either of the cell lines at both concentrations, whereas Complex substantially reduced viability that was comparable to doxorubicin at indicated concentration. Data are expressed as mean ± S.D., n = 3; * p
    Anti P53 Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology mouse anti p53
    PKCζ–PIASy association is critical for <t>p53</t> SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO – for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO − or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity ( Woo et al., 2008 ). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P
    Mouse Anti P53, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 754 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson p53
    Inhibition of Aurora B induces <t>p53</t> in a breast cancer xenograft model. ( A ) MCF7-Her18 cells were treated with increasing concentrations of AZD1152-HQPA as labeled for 48 h. Antigens for the immunoblots shown are labeled on the left. Mdm2 is indicated as full length (90 kDa) and cleavage product (60 kDa). ( B ) MCF7-Her18 xenografts from nude mice (indicated by mouse number) treated with AZD1152 were immunoblotted for p53 and p53 target genes. The protein expression of Bax, Puma, and Actin was quantified from Western blot films using Image J program. Bax/Actin and Puma/Actin ratios were calculated. Bar graphs and one-way ANOVA statistic analyses with Turkey test were done with GraphPad Prism 5.0c. ( C ) Representative photomicrographs are shown for immunohistochemical staining of p53 in MCF7-Her18 xenograft tumors treated with AZD1152. ( D ) Percentage p53-positive ( Upper ) and average p53 immunostaining intensity ( Lower ) from automated quantitative image analysis of immunohistochemical staining of MCF7-Her18 nude mouse xenografts treated with AZD1152. * P
    P53, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 680 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc antibodies against p53
    Inhibition of Aurora B induces <t>p53</t> in a breast cancer xenograft model. ( A ) MCF7-Her18 cells were treated with increasing concentrations of AZD1152-HQPA as labeled for 48 h. Antigens for the immunoblots shown are labeled on the left. Mdm2 is indicated as full length (90 kDa) and cleavage product (60 kDa). ( B ) MCF7-Her18 xenografts from nude mice (indicated by mouse number) treated with AZD1152 were immunoblotted for p53 and p53 target genes. The protein expression of Bax, Puma, and Actin was quantified from Western blot films using Image J program. Bax/Actin and Puma/Actin ratios were calculated. Bar graphs and one-way ANOVA statistic analyses with Turkey test were done with GraphPad Prism 5.0c. ( C ) Representative photomicrographs are shown for immunohistochemical staining of p53 in MCF7-Her18 xenograft tumors treated with AZD1152. ( D ) Percentage p53-positive ( Upper ) and average p53 immunostaining intensity ( Lower ) from automated quantitative image analysis of immunohistochemical staining of MCF7-Her18 nude mouse xenografts treated with AZD1152. * P
    Antibodies Against P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 623 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Agilent technologies anti p53
    Keratinizing moderately differentiated SCC revealed a moderate <t>p53</t> immunopositivity.
    Anti P53, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 94/100, based on 664 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore p53
    Proposed mechanism of the inhibitory process. The novel polysaccharide derived from algae extract upregulates the phosphorylation of JNK, activates the downstream cascades of <t>p53,</t> caspase-9 and caspase-3, and then leads to the inhibition of cancer cell proliferation, induces cell apoptosis and cell cycle arrest. By contrast, the polysaccharide does not affect the cancer cell migration, which is mediated through the p38 MAPK signaling pathway or the downstream MMP-9/MMP-2. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase.
    P53, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho p53 ser15
    Proposed mechanism of the inhibitory process. The novel polysaccharide derived from algae extract upregulates the phosphorylation of JNK, activates the downstream cascades of <t>p53,</t> caspase-9 and caspase-3, and then leads to the inhibition of cancer cell proliferation, induces cell apoptosis and cell cycle arrest. By contrast, the polysaccharide does not affect the cancer cell migration, which is mediated through the p38 MAPK signaling pathway or the downstream MMP-9/MMP-2. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase.
    Anti Phospho P53 Ser15, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The level of huntingtin protein is lower in mice deficient in p53

    Journal:

    Article Title: Genetic interaction between expanded murine Hdh alleles and p53 reveals deleterious effects of p53 on Huntington's disease pathogenesis

    doi: 10.1016/j.nbd.2006.08.002

    Figure Lengend Snippet: The level of huntingtin protein is lower in mice deficient in p53

    Article Snippet: p53 +/+ and p53 −/− MEFs were transiently transfected using Lipofectamine reagent (Invitrogen, Carlsbad, CA) with varying doses of p53 expression vector.

    Techniques: Mouse Assay

    p53 modulates huntingtin promoter activity and the level of huntingtin protein Mouse Hdh proximal promoter region A half consensus sequence corresponding to the p53 response element (shaded in gray) is located ∼1285bp upstream from translation

    Journal:

    Article Title: Genetic interaction between expanded murine Hdh alleles and p53 reveals deleterious effects of p53 on Huntington's disease pathogenesis

    doi: 10.1016/j.nbd.2006.08.002

    Figure Lengend Snippet: p53 modulates huntingtin promoter activity and the level of huntingtin protein Mouse Hdh proximal promoter region A half consensus sequence corresponding to the p53 response element (shaded in gray) is located ∼1285bp upstream from translation

    Article Snippet: p53 +/+ and p53 −/− MEFs were transiently transfected using Lipofectamine reagent (Invitrogen, Carlsbad, CA) with varying doses of p53 expression vector.

    Techniques: Activity Assay, Sequencing

    Loss of p53 partially rescues aspects of the HD phenotype The level of proenkephalin mRNA was determined by northern blot in the total brain of male mice at 80 (left panel), 140 (middle panel), or 180 days (right panel) of age. Blots were stripped and

    Journal:

    Article Title: Genetic interaction between expanded murine Hdh alleles and p53 reveals deleterious effects of p53 on Huntington's disease pathogenesis

    doi: 10.1016/j.nbd.2006.08.002

    Figure Lengend Snippet: Loss of p53 partially rescues aspects of the HD phenotype The level of proenkephalin mRNA was determined by northern blot in the total brain of male mice at 80 (left panel), 140 (middle panel), or 180 days (right panel) of age. Blots were stripped and

    Article Snippet: p53 +/+ and p53 −/− MEFs were transiently transfected using Lipofectamine reagent (Invitrogen, Carlsbad, CA) with varying doses of p53 expression vector.

    Techniques: Northern Blot, Mouse Assay

    Model of Hdh and p53 interaction Linear model of interaction : p53 resides upstream of Hdh and modifies the HD phentoype through the regulation of huntingtin level. Branched model of interaction : p53 resides upstream of Hdh and modifies the HD phentoype

    Journal:

    Article Title: Genetic interaction between expanded murine Hdh alleles and p53 reveals deleterious effects of p53 on Huntington's disease pathogenesis

    doi: 10.1016/j.nbd.2006.08.002

    Figure Lengend Snippet: Model of Hdh and p53 interaction Linear model of interaction : p53 resides upstream of Hdh and modifies the HD phentoype through the regulation of huntingtin level. Branched model of interaction : p53 resides upstream of Hdh and modifies the HD phentoype

    Article Snippet: p53 +/+ and p53 −/− MEFs were transiently transfected using Lipofectamine reagent (Invitrogen, Carlsbad, CA) with varying doses of p53 expression vector.

    Techniques:

    (A) The MYCN-ChIP-Seq database and p53-ChIP-Seq composite database were analyzed to determine binding patterns at the MYCN (E-box) and p53 (p53-RE) promoters in the top genes. The p53-repressed and p53-activated genes under low MYCN or high MYCN conditions were analyzed and summarized here for the presence or absence of E-box and p53-REs. Top 30 genes in each category by fold change were analyzed. Red= MYCN E-box present; Blue= p53-RE present. (B) Functional annotations of p53 response genes under high MYCN condition were analyzed and found to be highly correlated with worse prognosis and MYCN levels in NB patients. R2: Kosak (n=498) dataset of NB patient was used.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) The MYCN-ChIP-Seq database and p53-ChIP-Seq composite database were analyzed to determine binding patterns at the MYCN (E-box) and p53 (p53-RE) promoters in the top genes. The p53-repressed and p53-activated genes under low MYCN or high MYCN conditions were analyzed and summarized here for the presence or absence of E-box and p53-REs. Top 30 genes in each category by fold change were analyzed. Red= MYCN E-box present; Blue= p53-RE present. (B) Functional annotations of p53 response genes under high MYCN condition were analyzed and found to be highly correlated with worse prognosis and MYCN levels in NB patients. R2: Kosak (n=498) dataset of NB patient was used.

    Article Snippet: A total of 0.25 mg crude nuclear protein extract was incubated overnight at 4°C with 2 μg of anti-p53 (Ab-7, sheep, Calbiochem) or 2 μg of anti-FLAG antibodies (M2, Sigma Aldrich).

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Functional Assay

    Schematic model for impact of MYCN and p53 interactions on neuroblastoma tumor biology MYCN can complex with p53 via binding to the C-terminal domain when high levels of both MYCN and p53 are present in the nucleus as is the case when p53 wild type/MYCN amplified neuroblastoma is exposed to genotoxic damage. (A) Levels of free MYCN alter neuroblastoma responses to therapy (B) p53 transcriptional response is modified through changes in chromatin affinity and specificity. (C) Changes in DNA damage responses may promote mutation and drug resistance in MYCN amplified cancers.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: Schematic model for impact of MYCN and p53 interactions on neuroblastoma tumor biology MYCN can complex with p53 via binding to the C-terminal domain when high levels of both MYCN and p53 are present in the nucleus as is the case when p53 wild type/MYCN amplified neuroblastoma is exposed to genotoxic damage. (A) Levels of free MYCN alter neuroblastoma responses to therapy (B) p53 transcriptional response is modified through changes in chromatin affinity and specificity. (C) Changes in DNA damage responses may promote mutation and drug resistance in MYCN amplified cancers.

    Article Snippet: A total of 0.25 mg crude nuclear protein extract was incubated overnight at 4°C with 2 μg of anti-p53 (Ab-7, sheep, Calbiochem) or 2 μg of anti-FLAG antibodies (M2, Sigma Aldrich).

    Techniques: Binding Assay, Amplification, Modification, Mutagenesis

    (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.

    Article Snippet: A total of 0.25 mg crude nuclear protein extract was incubated overnight at 4°C with 2 μg of anti-p53 (Ab-7, sheep, Calbiochem) or 2 μg of anti-FLAG antibodies (M2, Sigma Aldrich).

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Negative Control, Western Blot, In Vitro, Expressing, Incubation, Staining, Purification, Recombinant, Transfection, Cell Culture, Amplification

    (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized onto glutathione-agarose beads. Input and pull-down samples were immunoblotted using anti-MYCN and anti-MAX antibodies. Stain-Free total protein staining was used as the loading control. (C) GST pull-down assay of MYCN truncations. Crude nuclear protein extract from transiently transfected p53-overexpressing HEK-293T cells was incubated with different MYCN-GST fragments immobilized on glutathione-agarose beads. GST alone was used as a negative control. Input and pull-down samples were immunoblotted using anti-p53 (DO-1) antibody. Ponceau staining was used as a loading control.

    Article Snippet: A total of 0.25 mg crude nuclear protein extract was incubated overnight at 4°C with 2 μg of anti-p53 (Ab-7, sheep, Calbiochem) or 2 μg of anti-FLAG antibodies (M2, Sigma Aldrich).

    Techniques: Construct, Activation Assay, Binding Assay, Clone Assay, Plasmid Preparation, Purification, Amplification, Incubation, Negative Control, Staining, Pull Down Assay, Transfection

    (A) RNA-Seq was performed on MYCN3 cells under different p53 and MYCN high conditions. Treatment conditions were compared to determine the effect of low and high MYCN levels on p53 response. p53 activated and repressed genes under high and low MYCN conditions were determined and shown with Venn diagrams. (B) Waterfall plots of the relative changes in gene expression of common genes shows the p53-regulated genes under low MYCN vs. high MYCN conditions. High MYCN levels relatively repressed the p53-up-regulated genes and relatively de-repressed the p53-down-regulated genes. (C, D) Functional annotation analysis of differentially expressed genes using the DAVID Bioinformatics platform. Representative genes analyzed are shown in boxes. (E) Gene Set Enrichment Analysis (GSEA) shows a statistically significant and robust interaction between the MYCN and p53 transcriptional programs. Genes induced by MYCN in either low or high p53 conditions are suppressed in the p53 transcriptional response regardless of MYCN levels (Normalized Enrichment Score [NES]

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: (A) RNA-Seq was performed on MYCN3 cells under different p53 and MYCN high conditions. Treatment conditions were compared to determine the effect of low and high MYCN levels on p53 response. p53 activated and repressed genes under high and low MYCN conditions were determined and shown with Venn diagrams. (B) Waterfall plots of the relative changes in gene expression of common genes shows the p53-regulated genes under low MYCN vs. high MYCN conditions. High MYCN levels relatively repressed the p53-up-regulated genes and relatively de-repressed the p53-down-regulated genes. (C, D) Functional annotation analysis of differentially expressed genes using the DAVID Bioinformatics platform. Representative genes analyzed are shown in boxes. (E) Gene Set Enrichment Analysis (GSEA) shows a statistically significant and robust interaction between the MYCN and p53 transcriptional programs. Genes induced by MYCN in either low or high p53 conditions are suppressed in the p53 transcriptional response regardless of MYCN levels (Normalized Enrichment Score [NES]

    Article Snippet: A total of 0.25 mg crude nuclear protein extract was incubated overnight at 4°C with 2 μg of anti-p53 (Ab-7, sheep, Calbiochem) or 2 μg of anti-FLAG antibodies (M2, Sigma Aldrich).

    Techniques: RNA Sequencing Assay, Expressing, Functional Assay

    Quantification of p53 and MYCN binding using ChIP-qPCR of p53 and MYCN target genes under different treatment conditions ) and primer binding locations and respective primer names are shown here with red arrows. MYCN- and p53-ChIP was performed with their respective antibodies as described in Methods. ChIP-qPCR with E-box or p53-RE primers were performed on DNA from both the MYCN-ChIP and p53 ChIP assays, and plotted as individual bar graphs. Cross-ChIP-qPCR experiments using E-box qPCR primers with p53-ChIP DNA or p53-RE primers with MYCN-ChIP DNA were also performed and shown here. The ChIP-qPCR and cross-ChIP-qPCR assays were performed in response to MYCN induction with doxycycline and p53 induction with either Nutlin-3a or with genotoxic chemotherapy treatments. MYCN3 cells were treated with low (10 μg/ml, +) or high (20 μg/ml, ++) doses of VP-16 in the presence or absence of doxycycline for the ChIP assays. D= Doxycycline, N=Nutlin-3a, V=VP-16. (A) CDKN1A (p21): primers p21-CP (p53-RE primer) and p21-CM (E-Box primer). ChIP-qPCR and Cross-ChIP-qPCR graphs for p21 locus. (B) A Re-ChIP assay was performed for p53 binding site on CDKN1A promoter. The p53 ChIP material was re-ChIPed using either IgG, p53 or MYCN antibody followed and analyzed by PCR amplification using p21-CP primers. The agarose gel is shown with proper Input and loading controls. (C) SESN1: primers SESN1-CP (p53-RE primer) and SESN1-CM (E-Box primer). ChIP-qPCR and Cross-ChIP-qPCR graphs for SESN1 locus. (D) CHEK1: primers CHEK1-CM (E-Box primer). ChIP-qPCR and ross-ChIP-qPCR graphs for CHEK1 locus. (E) CDC6: primers CDC6-CM (E-Box primer). ChIP-qPCR and Cross-ChIP-qPCR graphs for CDC6 locus. * p

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: Quantification of p53 and MYCN binding using ChIP-qPCR of p53 and MYCN target genes under different treatment conditions ) and primer binding locations and respective primer names are shown here with red arrows. MYCN- and p53-ChIP was performed with their respective antibodies as described in Methods. ChIP-qPCR with E-box or p53-RE primers were performed on DNA from both the MYCN-ChIP and p53 ChIP assays, and plotted as individual bar graphs. Cross-ChIP-qPCR experiments using E-box qPCR primers with p53-ChIP DNA or p53-RE primers with MYCN-ChIP DNA were also performed and shown here. The ChIP-qPCR and cross-ChIP-qPCR assays were performed in response to MYCN induction with doxycycline and p53 induction with either Nutlin-3a or with genotoxic chemotherapy treatments. MYCN3 cells were treated with low (10 μg/ml, +) or high (20 μg/ml, ++) doses of VP-16 in the presence or absence of doxycycline for the ChIP assays. D= Doxycycline, N=Nutlin-3a, V=VP-16. (A) CDKN1A (p21): primers p21-CP (p53-RE primer) and p21-CM (E-Box primer). ChIP-qPCR and Cross-ChIP-qPCR graphs for p21 locus. (B) A Re-ChIP assay was performed for p53 binding site on CDKN1A promoter. The p53 ChIP material was re-ChIPed using either IgG, p53 or MYCN antibody followed and analyzed by PCR amplification using p21-CP primers. The agarose gel is shown with proper Input and loading controls. (C) SESN1: primers SESN1-CP (p53-RE primer) and SESN1-CM (E-Box primer). ChIP-qPCR and Cross-ChIP-qPCR graphs for SESN1 locus. (D) CHEK1: primers CHEK1-CM (E-Box primer). ChIP-qPCR and ross-ChIP-qPCR graphs for CHEK1 locus. (E) CDC6: primers CDC6-CM (E-Box primer). ChIP-qPCR and Cross-ChIP-qPCR graphs for CDC6 locus. * p

    Article Snippet: A total of 0.25 mg crude nuclear protein extract was incubated overnight at 4°C with 2 μg of anti-p53 (Ab-7, sheep, Calbiochem) or 2 μg of anti-FLAG antibodies (M2, Sigma Aldrich).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    MYCN and p53 co-localize and bind to each other (A) The MYCN3 cell line was generated by transfecting a Tet-On plasmid containing full-length MYCN cDNA. MYCN3 cells were treated with Doxycycline to induce MYCN levels and with Nutlin-3a to induce p53 levels. Western blot showing MYCN and p53 protein levels under different treatment conditions. (B) Proximity ligation assays (PLA) for MYCN and p53 binding were performed using MYCN3 cells in the presence or absence of doxycycline and Nutlin-3a treatments. Additional controls were performed to determine antibody specificity. Representative images of control and combined doxycycline and Nutlin-3a treated cells are shown. A Scatter-plot and the mean ± SEM of the number of PLA spots per cell are shown in the bottom panel. The p -values of the difference between combination treatments and all other groups was

    Journal: Oncotarget

    Article Title: MYCN acts as a direct co-regulator of p53 in MYCN amplified neuroblastoma

    doi: 10.18632/oncotarget.24859

    Figure Lengend Snippet: MYCN and p53 co-localize and bind to each other (A) The MYCN3 cell line was generated by transfecting a Tet-On plasmid containing full-length MYCN cDNA. MYCN3 cells were treated with Doxycycline to induce MYCN levels and with Nutlin-3a to induce p53 levels. Western blot showing MYCN and p53 protein levels under different treatment conditions. (B) Proximity ligation assays (PLA) for MYCN and p53 binding were performed using MYCN3 cells in the presence or absence of doxycycline and Nutlin-3a treatments. Additional controls were performed to determine antibody specificity. Representative images of control and combined doxycycline and Nutlin-3a treated cells are shown. A Scatter-plot and the mean ± SEM of the number of PLA spots per cell are shown in the bottom panel. The p -values of the difference between combination treatments and all other groups was

    Article Snippet: A total of 0.25 mg crude nuclear protein extract was incubated overnight at 4°C with 2 μg of anti-p53 (Ab-7, sheep, Calbiochem) or 2 μg of anti-FLAG antibodies (M2, Sigma Aldrich).

    Techniques: Generated, Plasmid Preparation, Western Blot, Ligation, Proximity Ligation Assay, Binding Assay

    M induces nuclear localization of a p53 NLS mutant (p53 KRKKK ) and this is dependent on the nuclear localization signal of M and efficient interaction between p53 and the M protein. A. p53−/− HCT116 cells were transfected with the plasmids indicated. Localization of full-length p53 was determined by immunofluorescence. B. Quantification of immunofluorescence staining in A. 300 cells per transfection condition were scored as having primarily nuclear p53, primarily cytoplasmic p53, or both nuclear and cytoplasmic p53. Values are the average of three independent experiments and error bars represent standard error. * p

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: M induces nuclear localization of a p53 NLS mutant (p53 KRKKK ) and this is dependent on the nuclear localization signal of M and efficient interaction between p53 and the M protein. A. p53−/− HCT116 cells were transfected with the plasmids indicated. Localization of full-length p53 was determined by immunofluorescence. B. Quantification of immunofluorescence staining in A. 300 cells per transfection condition were scored as having primarily nuclear p53, primarily cytoplasmic p53, or both nuclear and cytoplasmic p53. Values are the average of three independent experiments and error bars represent standard error. * p

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: Mutagenesis, Transfection, Immunofluorescence, Staining

    The M protein does not enhance p53 stability by disrupting MDM2 function. p53 null Saos-2 cells were transfected with combinations of p53, M, and MDM2 as indicated. A. The M protein increases MDM2-p53 interactions. Lysates from transfected Saos-2 cells were immunoprecipitated with a p53 antibody, and Western blots were probed with antibodies to p53 and MDM2. MDM2 is readily detected in complex with p53 in the presence of M. The Western blot of total lysate prior to immunoprecipitation is also shown. B. Ubiquitination of p53. U2OS cells were transfected with the indicated combinations of M and MDM2. Total p53 was immunoprecipitated and Western blots were probed with antibodies to Ubiquitin, p53, and MDM2. IP: Immunoprecipitate, WB: Western blot, WCE: whole cell extract, Ub: ubiquitin.

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: The M protein does not enhance p53 stability by disrupting MDM2 function. p53 null Saos-2 cells were transfected with combinations of p53, M, and MDM2 as indicated. A. The M protein increases MDM2-p53 interactions. Lysates from transfected Saos-2 cells were immunoprecipitated with a p53 antibody, and Western blots were probed with antibodies to p53 and MDM2. MDM2 is readily detected in complex with p53 in the presence of M. The Western blot of total lysate prior to immunoprecipitation is also shown. B. Ubiquitination of p53. U2OS cells were transfected with the indicated combinations of M and MDM2. Total p53 was immunoprecipitated and Western blots were probed with antibodies to Ubiquitin, p53, and MDM2. IP: Immunoprecipitate, WB: Western blot, WCE: whole cell extract, Ub: ubiquitin.

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: Transfection, Immunoprecipitation, Western Blot

    The M protein enhances the stability of full-length p53. U2OS cells were transfected with plasmids encoding M and M mutants as indicated. A. p53 protein levels in the absence of de novo protein synthesis. Cells were treated with cycloheximide and protein lysates collected at the indicated time points followed by a Western blot for total p53. B. Graphical representation of total p53 levels in A after normalization to actin levels. Blots were scanned on a GE Storm 860 imager and quantitated using ImageQuant software. The graphs indicate the percent p53 remaining relative to the zero time point. Values represent the average of three independent experiments and error bars represent standard error. Statistical analyses (ANOVA) indicated that empty vector transfected U2OS cells exhibited significantly less p53 stability than cells transfected with M or mutant M vectors (P

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: The M protein enhances the stability of full-length p53. U2OS cells were transfected with plasmids encoding M and M mutants as indicated. A. p53 protein levels in the absence of de novo protein synthesis. Cells were treated with cycloheximide and protein lysates collected at the indicated time points followed by a Western blot for total p53. B. Graphical representation of total p53 levels in A after normalization to actin levels. Blots were scanned on a GE Storm 860 imager and quantitated using ImageQuant software. The graphs indicate the percent p53 remaining relative to the zero time point. Values represent the average of three independent experiments and error bars represent standard error. Statistical analyses (ANOVA) indicated that empty vector transfected U2OS cells exhibited significantly less p53 stability than cells transfected with M or mutant M vectors (P

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: Transfection, Western Blot, Software, Plasmid Preparation, Mutagenesis

    Effects of wild-type p53, mutant p53, and the p53 m allele on growth suppression of human osteosarcoma cells. A-C. Colony formation in osteosarcoma cells. Saos-2 cells (null for p53) (A), U2OS cells (wild-type for p53) (B), and TE85 (containing mutant p53) (C), were transfected with empty vector (zeocin resistance) constructs, wild-type p53 (zeocin resistance) expression constructs, and m allele expression (zeocin resistance) constructs. Forty-eight hours after transfection, osteosarcoma cells were selected in zeocin for two weeks and zeocin resistant colonies fixed, stained and counted. D. Saos-2 cells were transfected with a zeocin resistance gene construct expressing either no insert (empty vector), a mutant version of p53 (codon 172 arg→his), wild-type p53, or the truncated p53 m allele, or the indicated combination of vectors. Zeocin resistant colonies were identified and counted as described for panels A-C.

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: Effects of wild-type p53, mutant p53, and the p53 m allele on growth suppression of human osteosarcoma cells. A-C. Colony formation in osteosarcoma cells. Saos-2 cells (null for p53) (A), U2OS cells (wild-type for p53) (B), and TE85 (containing mutant p53) (C), were transfected with empty vector (zeocin resistance) constructs, wild-type p53 (zeocin resistance) expression constructs, and m allele expression (zeocin resistance) constructs. Forty-eight hours after transfection, osteosarcoma cells were selected in zeocin for two weeks and zeocin resistant colonies fixed, stained and counted. D. Saos-2 cells were transfected with a zeocin resistance gene construct expressing either no insert (empty vector), a mutant version of p53 (codon 172 arg→his), wild-type p53, or the truncated p53 m allele, or the indicated combination of vectors. Zeocin resistant colonies were identified and counted as described for panels A-C.

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: Mutagenesis, Transfection, Plasmid Preparation, Construct, Expressing, Staining

    The M protein localizes to the nucleus and interacts with wild-type p53. A. Diagram of human p53 and the M protein. Residues that were mutated to alanine in the NLS and tetramerization domain are shown in bold. AD: Activation Domain. DBD: DNA Binding Domain. NLS/TD: Nuclear Localization Signal/Tetramerization Domain. B. Localization of M and M mutants. Cells were transfected with GFP tagged M (panels a-c), GFP tagged M KRKKK (panels d-f), or GFP tagged M 348/350A (panels g-i) as indicated. The localization of M was determined by immunofluorescence and nuclei visualized by DAPI staining. 300 cells were counted and scored as having GFP-M mostly nuclear (N), mostly cytoplasmic (C), or both nuclear and cytoplasmic (B). Percentages listed are an average of three independent experiments. C. Interaction between p53 and M mutants. p53−/− HCT116 cells were transfected with GFP tagged p53 and M expression constructs as indicated. p53 was immunoprecipitated with an antibody specific to the N-terminus of p53. Membranes were probed with an antibody to GFP to detect both full-length p53 and the M protein.

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: The M protein localizes to the nucleus and interacts with wild-type p53. A. Diagram of human p53 and the M protein. Residues that were mutated to alanine in the NLS and tetramerization domain are shown in bold. AD: Activation Domain. DBD: DNA Binding Domain. NLS/TD: Nuclear Localization Signal/Tetramerization Domain. B. Localization of M and M mutants. Cells were transfected with GFP tagged M (panels a-c), GFP tagged M KRKKK (panels d-f), or GFP tagged M 348/350A (panels g-i) as indicated. The localization of M was determined by immunofluorescence and nuclei visualized by DAPI staining. 300 cells were counted and scored as having GFP-M mostly nuclear (N), mostly cytoplasmic (C), or both nuclear and cytoplasmic (B). Percentages listed are an average of three independent experiments. C. Interaction between p53 and M mutants. p53−/− HCT116 cells were transfected with GFP tagged p53 and M expression constructs as indicated. p53 was immunoprecipitated with an antibody specific to the N-terminus of p53. Membranes were probed with an antibody to GFP to detect both full-length p53 and the M protein.

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: Activation Assay, Binding Assay, Transfection, Immunofluorescence, Staining, Expressing, Construct, Immunoprecipitation

    The M protein induces nuclear accumulation of p53. U2OS cells were transfected with empty vector (Ev) or a plasmid encoding M. Cells were irradiated with 5 Grays of ionizing radiation or mock irradiated four hours prior to fixing and immunostaining for full-length p53. Top: Empty vector transfected cells. Middle: Empty vector transfected cells 4 hours following irradiation. Bottom: M transfected cells. Cells that are transfected with M (indicated by white arrows) have increased nuclear p53 compared to untransfected cells.

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: The M protein induces nuclear accumulation of p53. U2OS cells were transfected with empty vector (Ev) or a plasmid encoding M. Cells were irradiated with 5 Grays of ionizing radiation or mock irradiated four hours prior to fixing and immunostaining for full-length p53. Top: Empty vector transfected cells. Middle: Empty vector transfected cells 4 hours following irradiation. Bottom: M transfected cells. Cells that are transfected with M (indicated by white arrows) have increased nuclear p53 compared to untransfected cells.

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: Transfection, Plasmid Preparation, Irradiation, Immunostaining

    p53 protein levels are more stable in p53+/m MEFs compared to p53+/− MEFs. A. p53 protein levels in the absence of de novo protein synthesis. Low passage MEFs were treated with cycloheximide and cell lysates collected at the indicated time points. B. p53+/m MEFs exhibit increased p53 stability compared to p53+/− MEFs. Western blots were normalized to actin, quantitated by densitometry and graphed as the percent p53 remaining over time relative to the zero time point. The graph represents the average of three independent experiments (and independent MEF lines). Error bars represent standard error of the mean. Statistical analyses (ANOVA) indicated that cycloheximide treated p53+/m cells exhibited significant increases in p53 stability compared to similarly treated p53+/− cells (P = 0.04). C. p53 protein levels in p53+/m and p53+/− MEFs after treatment with proteasome inhibitors. Two different isolates of of low passage p53+/− MEFs (MEF-1: lanes 1,2; MEF-2: lanes 3,4) and p53+/m MEFs (MEF-3: lanes 5,6; MEF-4: lanes 7,8) were untreated or treated with proteasome inhibitors MG101 and MG132 for 6 hours prior to harvest. p53 protein levels accumulate upon proteasome inhibitor treatment in p53+/− MEFs, but are not significantly increased in p53+/m MEFs implying that p53 is protected from proteasomal degradation in p53+/m MEFs. D. p53 protein levels are less sensitive to proteasome inhibitors in p53+/m MEFs. Western blots in C were normalized to actin and quantitated by densitometry. The values shown are the average p53 levels of three independent MEF lines for each genotype and error bars represent standard error. *P

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: p53 protein levels are more stable in p53+/m MEFs compared to p53+/− MEFs. A. p53 protein levels in the absence of de novo protein synthesis. Low passage MEFs were treated with cycloheximide and cell lysates collected at the indicated time points. B. p53+/m MEFs exhibit increased p53 stability compared to p53+/− MEFs. Western blots were normalized to actin, quantitated by densitometry and graphed as the percent p53 remaining over time relative to the zero time point. The graph represents the average of three independent experiments (and independent MEF lines). Error bars represent standard error of the mean. Statistical analyses (ANOVA) indicated that cycloheximide treated p53+/m cells exhibited significant increases in p53 stability compared to similarly treated p53+/− cells (P = 0.04). C. p53 protein levels in p53+/m and p53+/− MEFs after treatment with proteasome inhibitors. Two different isolates of of low passage p53+/− MEFs (MEF-1: lanes 1,2; MEF-2: lanes 3,4) and p53+/m MEFs (MEF-3: lanes 5,6; MEF-4: lanes 7,8) were untreated or treated with proteasome inhibitors MG101 and MG132 for 6 hours prior to harvest. p53 protein levels accumulate upon proteasome inhibitor treatment in p53+/− MEFs, but are not significantly increased in p53+/m MEFs implying that p53 is protected from proteasomal degradation in p53+/m MEFs. D. p53 protein levels are less sensitive to proteasome inhibitors in p53+/m MEFs. Western blots in C were normalized to actin and quantitated by densitometry. The values shown are the average p53 levels of three independent MEF lines for each genotype and error bars represent standard error. *P

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: Western Blot

    Growth kinetics, cell cycle checkpoint control, and suppression of recombination of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. A. Representative growth kinetics of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. 750,000 cells were plated onto 10cm dishes and collected and counted on the days indicated. T-tests on multiple growth experiments indicated no significant difference between p53+/+ and p53+/m growth rates (P = 0.61), while growth rate differences between p53+/m and p53+/− MEFs were significantly different (P = 0.003). B. S phase fractions in rapidly dividing MEFs of different p53 genotypes. MEFs were analyzed for DNA content and BrdU incorporation by flow cytometry and the percentage of cells in S phase was calculated. T-tests indicated the S phase fractions of p53+/m and p53+/+ MEFs were not significantly different (P = 0.47), while S phase fractions of p53+/m and p53+/− MEFs were significantly different and are indicated by an asterisk (P = 0.05). C. Suppression of recombination in p53+/+, p53+/−, p53+/m, and p53−/− passage 1−3 MEFs. p53+/+, p53+/−, p53+/m, and p53−/− MEFs were infected with a retrovirus expressing two tandem copies of mutant forms of a GFP-Zeocin gene as well as a neomycin resistance marker. MEFs were selected with G418 or G418 plus Zeocin to determine the recombination frequencies. The p53+/m MEFs have a recombination frequency roughly equal to that of p53+/+ MEFs. The difference in recombination frequency between p53+/m and p53+/− MEFs is highly significant (P

    Journal: Mechanisms of ageing and development

    Article Title: Aging-Associated Truncated Form of p53 Interacts with Wild-Type p53 and Alters p53 Stability, Localization, and Activity

    doi: 10.1016/j.mad.2007.10.011

    Figure Lengend Snippet: Growth kinetics, cell cycle checkpoint control, and suppression of recombination of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. A. Representative growth kinetics of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. 750,000 cells were plated onto 10cm dishes and collected and counted on the days indicated. T-tests on multiple growth experiments indicated no significant difference between p53+/+ and p53+/m growth rates (P = 0.61), while growth rate differences between p53+/m and p53+/− MEFs were significantly different (P = 0.003). B. S phase fractions in rapidly dividing MEFs of different p53 genotypes. MEFs were analyzed for DNA content and BrdU incorporation by flow cytometry and the percentage of cells in S phase was calculated. T-tests indicated the S phase fractions of p53+/m and p53+/+ MEFs were not significantly different (P = 0.47), while S phase fractions of p53+/m and p53+/− MEFs were significantly different and are indicated by an asterisk (P = 0.05). C. Suppression of recombination in p53+/+, p53+/−, p53+/m, and p53−/− passage 1−3 MEFs. p53+/+, p53+/−, p53+/m, and p53−/− MEFs were infected with a retrovirus expressing two tandem copies of mutant forms of a GFP-Zeocin gene as well as a neomycin resistance marker. MEFs were selected with G418 or G418 plus Zeocin to determine the recombination frequencies. The p53+/m MEFs have a recombination frequency roughly equal to that of p53+/+ MEFs. The difference in recombination frequency between p53+/m and p53+/− MEFs is highly significant (P

    Article Snippet: As the M protein contains the oligomerization domain of p53, we next examined the ability of the M protein to interact with full-length p53 ( ). p53−/− HCT116 cells were transfected with p53 and M as indicated and p53 immunoprecipitated with an antibody specific to the N-terminal domain of p53 (DO-1, Santa Cruz) to examine the interaction between p53 and M. The M protein retains the ability to bind to full-length, wild-type p53 ( , lane 7).

    Techniques: BrdU Incorporation Assay, Flow Cytometry, Cytometry, Infection, Expressing, Mutagenesis, Marker

    PON2 reduces the IGF-1 level via C-Jun transcription factor in ID8 cells. ID8 EV and ID8 hPON2 cell culture lysates were prepared and utilized for chromatin immunoprecipitation assays with C-Jun ( a ) and p53 ( b ) antibodies. c) Equal number of ID8 hPON2 cells were plated in six-well plates. At 60 % confluence level, ID8 hPON2 cells were transfected with either hPON2 siRNA or scrambled siRNA, and PON2 expression analyzed by western blotting. d) Data in c are quantified using ImageJ software. e) Four six-well plates of cells were pooled from PON2-siRNA-treated ID8 hPON2 or scrambled siRNA-treated ID8 hPON2 cells. Chromatin immunoprecipitation assay was performed and C-Jun promoter was quantified ( e ) as described in the materials and methods section. Cell culture supernatants obtained from cells described under ( e ) and IGF-1 level was analyzed by ELISA ( f ), g ) cell proliferation, and h) mitochondrial superoxide levels were measured as described in the method section. * p

    Journal: Cell Death & Disease

    Article Title: Paraoxonase 2 overexpression inhibits tumor development in a mouse model of ovarian cancer

    doi: 10.1038/s41419-018-0395-2

    Figure Lengend Snippet: PON2 reduces the IGF-1 level via C-Jun transcription factor in ID8 cells. ID8 EV and ID8 hPON2 cell culture lysates were prepared and utilized for chromatin immunoprecipitation assays with C-Jun ( a ) and p53 ( b ) antibodies. c) Equal number of ID8 hPON2 cells were plated in six-well plates. At 60 % confluence level, ID8 hPON2 cells were transfected with either hPON2 siRNA or scrambled siRNA, and PON2 expression analyzed by western blotting. d) Data in c are quantified using ImageJ software. e) Four six-well plates of cells were pooled from PON2-siRNA-treated ID8 hPON2 or scrambled siRNA-treated ID8 hPON2 cells. Chromatin immunoprecipitation assay was performed and C-Jun promoter was quantified ( e ) as described in the materials and methods section. Cell culture supernatants obtained from cells described under ( e ) and IGF-1 level was analyzed by ELISA ( f ), g ) cell proliferation, and h) mitochondrial superoxide levels were measured as described in the method section. * p

    Article Snippet: The chromatin was further immunoprecipitated with C-Jun (#9165), p53 (#2524) (Cell signaling technology, Danvers, MA), and rabbit or mouse normal IgG as a control.

    Techniques: Cell Culture, Chromatin Immunoprecipitation, Transfection, Expressing, Western Blot, Software, Enzyme-linked Immunosorbent Assay

    Genotypes of Control Embryos in Figures and the Genders Associated with Phenotypes (a) Table identifying the genotypes of control embryos shown for each analysis. (b) The table shows the number of male and female p53 25,26,53,54/+ embryos observed with the indicated phenotypes, as assessed by Zfy PCR. Phenotypes are well represented in both sexes.

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: Genotypes of Control Embryos in Figures and the Genders Associated with Phenotypes (a) Table identifying the genotypes of control embryos shown for each analysis. (b) The table shows the number of male and female p53 25,26,53,54/+ embryos observed with the indicated phenotypes, as assessed by Zfy PCR. Phenotypes are well represented in both sexes.

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Polymerase Chain Reaction

    p53 25,26,53,54 Interacts with and Increases Wild-Type p53 Levels and Activity (a) Immunoblot for total p53 (top row) and wild-type (WT) p53 (2 nd row) in untreated and doxorubicin (Dox)-treated MEFs. Actin loading control. (b) Anti-FLAG immunoprecipitation from p53 −/− MEFs transiently overexpressing HA-p53 and FLAG-p53 or FLAG-p53 25,26,53,54 . Negative controls: HA-MBP, FLAG-eGFP. *See Extended-Data Figure 7b (c) Immunoblot of p53 −/− MEFs transiently overexpressing HA-p53 and increasing amounts of FLAG-p53 or FLAG-p53 25,26,53,54 . Bottom: Average+/−s.d. HA-p53 protein levels relative to lane 1 and normalized to Actin. (n=3). (d) p53 target gene expression in untreated MEFs. Averages+/−s.d. normalized to β-actin . (n=4) *,***p-values

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54 Interacts with and Increases Wild-Type p53 Levels and Activity (a) Immunoblot for total p53 (top row) and wild-type (WT) p53 (2 nd row) in untreated and doxorubicin (Dox)-treated MEFs. Actin loading control. (b) Anti-FLAG immunoprecipitation from p53 −/− MEFs transiently overexpressing HA-p53 and FLAG-p53 or FLAG-p53 25,26,53,54 . Negative controls: HA-MBP, FLAG-eGFP. *See Extended-Data Figure 7b (c) Immunoblot of p53 −/− MEFs transiently overexpressing HA-p53 and increasing amounts of FLAG-p53 or FLAG-p53 25,26,53,54 . Bottom: Average+/−s.d. HA-p53 protein levels relative to lane 1 and normalized to Actin. (n=3). (d) p53 target gene expression in untreated MEFs. Averages+/−s.d. normalized to β-actin . (n=4) *,***p-values

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Activity Assay, Immunoprecipitation, Expressing

    Model for Examining p53-Associated Developmental Phenotypes (a) Schematic of p53 25,26 , p53 53,54 , and p53 25,26,53,54 mutant p53 proteins. TAD: Transactivation Domain 1 or 2, PRD: Proline-Rich Domain, Tet: Tetramerization Domain, Basic: Basic Residue-Rich Domain. (b) p53 LSL-mut/+ mice (where mut can denote any of the p53 TAD mutants) were crossed to p53 +/+ ;CMV-Cre mice, which express Cre in the germline, to assess viability and developmental phenotypes of the p53 mutant-expressing progeny. (c) Table summarizing the actual genotypes and ultimate functional genotypes of progeny from crosses of p53 LSL-25,26,53,54/+ and p53 +/− ;CMV-Cre mice, as used throughout the manuscript. While p53 LSL-25,26,53,54/+ ; CMV-Cre is the actual initial genotype, when Cre acts to delete the Lox-Stop-Lox cassette, the genotype will be written as p53 25,26,53,54/+ to reflect this recombination. In the text and figure labels, the Cre nomenclature for both control and p53 25,26,53,54/+ embryos is excluded for simplicity. Controls for analyses comprise embryos both with and without the CMV-Cre transgene, as summarized in Extended-Data Fig. 3 .

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: Model for Examining p53-Associated Developmental Phenotypes (a) Schematic of p53 25,26 , p53 53,54 , and p53 25,26,53,54 mutant p53 proteins. TAD: Transactivation Domain 1 or 2, PRD: Proline-Rich Domain, Tet: Tetramerization Domain, Basic: Basic Residue-Rich Domain. (b) p53 LSL-mut/+ mice (where mut can denote any of the p53 TAD mutants) were crossed to p53 +/+ ;CMV-Cre mice, which express Cre in the germline, to assess viability and developmental phenotypes of the p53 mutant-expressing progeny. (c) Table summarizing the actual genotypes and ultimate functional genotypes of progeny from crosses of p53 LSL-25,26,53,54/+ and p53 +/− ;CMV-Cre mice, as used throughout the manuscript. While p53 LSL-25,26,53,54/+ ; CMV-Cre is the actual initial genotype, when Cre acts to delete the Lox-Stop-Lox cassette, the genotype will be written as p53 25,26,53,54/+ to reflect this recombination. In the text and figure labels, the Cre nomenclature for both control and p53 25,26,53,54/+ embryos is excluded for simplicity. Controls for analyses comprise embryos both with and without the CMV-Cre transgene, as summarized in Extended-Data Fig. 3 .

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Mouse Assay, Expressing, Functional Assay

    p53 25,26,53,54/− Mice, but not p53 25,26/+ or p53 25,26/− Mice, are Viable (a) Crosses of p53 LSL-25,26/+ with p53 +/+ ;CMV-Cre or p53 +/− ;CMV-Cre mice reveal a decrease in viable pups expressing p53 25,26 at E9.5-E10.5. Observed numbers of live and dead pups compared to the expected numbers of live pups are indicated. [Observed (Expected)] The genotypes of p53 25,26/+ and p53 25,26/− mice carrying a CMV-Cre transgene lack the LSL designation because the Lox-Stop-Lox element has been deleted from the genome. Significance as assessed by Binomial distribution statistical tests on live pups: p=0.18 and 0.09 (b) Crosses of p53 LSL-25,26/+ or p53 LSL-25,26,53,54/+ with p53 +/− ;CMV-Cre mice reveal that p53 25,26,53,54/− mice, but not p53 25,26/− mice, are viable as assessed at postnatal day 21 (P21). Mut denotes either mutant allele. Observed numbers of pups compared to the expected numbers of pups are indicated. [Observed (Expected)] The genotypes of p53 mut/+ and p53 mut/− mice carrying a CMV-Cre transgene lack the LSL designation because the Lox-Stop-Lox element has been deleted from the genome. Lack of pups is significant at P21 as assessed by Binomial distribution statistical tests on live pups: p53 25,26/+ and p53 25,26/− : *p

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54/− Mice, but not p53 25,26/+ or p53 25,26/− Mice, are Viable (a) Crosses of p53 LSL-25,26/+ with p53 +/+ ;CMV-Cre or p53 +/− ;CMV-Cre mice reveal a decrease in viable pups expressing p53 25,26 at E9.5-E10.5. Observed numbers of live and dead pups compared to the expected numbers of live pups are indicated. [Observed (Expected)] The genotypes of p53 25,26/+ and p53 25,26/− mice carrying a CMV-Cre transgene lack the LSL designation because the Lox-Stop-Lox element has been deleted from the genome. Significance as assessed by Binomial distribution statistical tests on live pups: p=0.18 and 0.09 (b) Crosses of p53 LSL-25,26/+ or p53 LSL-25,26,53,54/+ with p53 +/− ;CMV-Cre mice reveal that p53 25,26,53,54/− mice, but not p53 25,26/− mice, are viable as assessed at postnatal day 21 (P21). Mut denotes either mutant allele. Observed numbers of pups compared to the expected numbers of pups are indicated. [Observed (Expected)] The genotypes of p53 mut/+ and p53 mut/− mice carrying a CMV-Cre transgene lack the LSL designation because the Lox-Stop-Lox element has been deleted from the genome. Lack of pups is significant at P21 as assessed by Binomial distribution statistical tests on live pups: p53 25,26/+ and p53 25,26/− : *p

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Mouse Assay, Expressing, Mutagenesis

    p53 Heterozygosity Partially Rescues Chd7-Null Embryos (a) qRT-PCR analysis of Chd7 in untreated MEFs derived from E13.5 p53 +/− and p53 25,26,53,54/+ embryos. Graphs indicate averages from four independent MEF lines, +/−s.d., after normalization to β-actin . ns=non-significant. (b) Left: Schematic of neural crest cell differentiation. Right: Representative qRT-PCR analysis of neural crest cell markers in neural crest-like cells differentiated from Chd7 +/+ and Chd7 −/− ( whi/whi ) mouse embryonic stem cells normalized to β-actin and compared to matched embryonic stem cells. (c) H E-stained E10.5 Chd7 +/+ p53 +/− (control), Chd7 −/− p53 +/+ , and Chd7 −/− p53 +/− embryos. The Chd7 −/− p53 +/+ embryo shown is necrotic as evidenced by cellular autolysis. (d) Close-up image of heart region, denoted by red box in panel c, in E10.5 Chd7 +/+ p53 +/− (control) and Chd7 −/− p53 +/− embryos.

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 Heterozygosity Partially Rescues Chd7-Null Embryos (a) qRT-PCR analysis of Chd7 in untreated MEFs derived from E13.5 p53 +/− and p53 25,26,53,54/+ embryos. Graphs indicate averages from four independent MEF lines, +/−s.d., after normalization to β-actin . ns=non-significant. (b) Left: Schematic of neural crest cell differentiation. Right: Representative qRT-PCR analysis of neural crest cell markers in neural crest-like cells differentiated from Chd7 +/+ and Chd7 −/− ( whi/whi ) mouse embryonic stem cells normalized to β-actin and compared to matched embryonic stem cells. (c) H E-stained E10.5 Chd7 +/+ p53 +/− (control), Chd7 −/− p53 +/+ , and Chd7 −/− p53 +/− embryos. The Chd7 −/− p53 +/+ embryo shown is necrotic as evidenced by cellular autolysis. (d) Close-up image of heart region, denoted by red box in panel c, in E10.5 Chd7 +/+ p53 +/− (control) and Chd7 −/− p53 +/− embryos.

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Quantitative RT-PCR, Derivative Assay, Cell Differentiation, Staining

    p53 25,26,53,54/+ Embryos Exhibit Additional Features of CHARGE Syndrome and p53-Dependent Cellular Responses (a) Double outlet right ventricle (DORV) in E13.5 p53 25,26,53,54/+ heart (50%, n=6). Top: Main pulmonary artery (MPA) connects via pulmonary valve (PV) to right ventricle (RV) in both control and p53 25,26,53,54/+ embryo. Bottom: Aorta (Ao) in control embryo connects to left ventricle (LV) via aortic valve (AV) Φ . Aorta in p53 25,26,53,54/+ embryo connects to RV via AV*. (b) Abnormal atrioventricular cushions in E13.5 p53 25,26,53,54/+ heart (75%, n=4) fail to elongateinto mature mitral (mv, arrowhead) and tricuspid (tv, arrow) valves. RA: right atrium; LA: left atrium. (c) E13.5 p53 25,26,53,54/+ kidneys are smaller (79%), with fewer average glomeruli (13 vs. 3; n=5; arrows), than controls. (d) p53 25,26,53,54/+ embryonic phenotypes observed in CHARGE (+present, −absent). (e) Left: Cleaved-caspase 3 (CC3; Top) and p53 (Bottom) immunohistochemistry in E15.5 retinas. Arrows: CC3-positive cells. Right: CC3-positive cells per retinal area. ***p-value=0.007; one-tailed Welsh’s t-test (n=5). (f) BrdU immunofluorescence in E9.5 Pax3 + NCCs (delineated by green-dotted line; Extended-Data Fig. 6c ). Right: Percentage BrdU-positive cells per total Pax3 + NCCs ***p-value=0.004 one-tailed Student’s t-test (n=4).

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54/+ Embryos Exhibit Additional Features of CHARGE Syndrome and p53-Dependent Cellular Responses (a) Double outlet right ventricle (DORV) in E13.5 p53 25,26,53,54/+ heart (50%, n=6). Top: Main pulmonary artery (MPA) connects via pulmonary valve (PV) to right ventricle (RV) in both control and p53 25,26,53,54/+ embryo. Bottom: Aorta (Ao) in control embryo connects to left ventricle (LV) via aortic valve (AV) Φ . Aorta in p53 25,26,53,54/+ embryo connects to RV via AV*. (b) Abnormal atrioventricular cushions in E13.5 p53 25,26,53,54/+ heart (75%, n=4) fail to elongateinto mature mitral (mv, arrowhead) and tricuspid (tv, arrow) valves. RA: right atrium; LA: left atrium. (c) E13.5 p53 25,26,53,54/+ kidneys are smaller (79%), with fewer average glomeruli (13 vs. 3; n=5; arrows), than controls. (d) p53 25,26,53,54/+ embryonic phenotypes observed in CHARGE (+present, −absent). (e) Left: Cleaved-caspase 3 (CC3; Top) and p53 (Bottom) immunohistochemistry in E15.5 retinas. Arrows: CC3-positive cells. Right: CC3-positive cells per retinal area. ***p-value=0.007; one-tailed Welsh’s t-test (n=5). (f) BrdU immunofluorescence in E9.5 Pax3 + NCCs (delineated by green-dotted line; Extended-Data Fig. 6c ). Right: Percentage BrdU-positive cells per total Pax3 + NCCs ***p-value=0.004 one-tailed Student’s t-test (n=4).

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Immunohistochemistry, One-tailed Test, Immunofluorescence

    p53 is Activated Upon Chd7 Deficiency and Contributes to Chd7 -null Phenotypes (a) p53 target gene expression in Chd7 +/+ and Chd7 −/− ( whi/whi ) NCCs. Averages+/−s.e.m. normalized to β-actin (n=5). *,**p-values

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 is Activated Upon Chd7 Deficiency and Contributes to Chd7 -null Phenotypes (a) p53 target gene expression in Chd7 +/+ and Chd7 −/− ( whi/whi ) NCCs. Averages+/−s.e.m. normalized to β-actin (n=5). *,**p-values

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Expressing

    p53 25,26,53,54 is Transactivation-Dead but Augments Wild-Type p53 Activity (a) Western blot analysis of p53 protein levels in untreated or doxorubicin-treated (0.2 μg/ml Dox) p53 −/− , p53 +/− , p53 25,26,53,54/− , and p53 25,26,53,54/+ MEFs. Actin serves as a loading control. (b) Western blot analysis of anti-FLAG immunoprecipitation from p53 −/− MEFs transiently overexpressing HA-p53 and FLAG-p53 or FLAG-p53 25,26,53,54 . HA-MBP and FLAG-eGFP were used as negative controls. Immunoprecipitated protein and 10% input were probed with either anti-HA or anti-FLAG. (μg ratio of HA-p53 to FLAG-p53 or FLAG-p53 25,26,53,54 plasmid DNA: 1:1 or 1:2.5). (Supplement to Fig. 3b ) (c) Heat map examining the transactivation capacity of p53 25,26,53,54 on p53-dependent genes identified by microarray analysis through comparison of six HrasV12;p53 wild-type mouse embryo fibroblast (MEF) lines to six HrasV12;p53 − null MEF lines, as previously described 3 . Three independent HrasV12;p53 25,26,53,54/25,26,53,54 MEFs lines were analyzed, and showed that the gene expression profiles were indistinguishable from HrasV12;p53 null cells. Numbered columns indicate independent MEF lines. Blue – repressed genes; Red – induced genes. (d) qRT-PCR analysis of p53 target gene expression in untreated MEFs derived from p53 +/+ and p53 25,26,53,54/+ E13.5 embryos. Graphs indicate averages from four independent MEF lines, +/−SD, after normalization to β-actin . **,*** denote p-values of

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54 is Transactivation-Dead but Augments Wild-Type p53 Activity (a) Western blot analysis of p53 protein levels in untreated or doxorubicin-treated (0.2 μg/ml Dox) p53 −/− , p53 +/− , p53 25,26,53,54/− , and p53 25,26,53,54/+ MEFs. Actin serves as a loading control. (b) Western blot analysis of anti-FLAG immunoprecipitation from p53 −/− MEFs transiently overexpressing HA-p53 and FLAG-p53 or FLAG-p53 25,26,53,54 . HA-MBP and FLAG-eGFP were used as negative controls. Immunoprecipitated protein and 10% input were probed with either anti-HA or anti-FLAG. (μg ratio of HA-p53 to FLAG-p53 or FLAG-p53 25,26,53,54 plasmid DNA: 1:1 or 1:2.5). (Supplement to Fig. 3b ) (c) Heat map examining the transactivation capacity of p53 25,26,53,54 on p53-dependent genes identified by microarray analysis through comparison of six HrasV12;p53 wild-type mouse embryo fibroblast (MEF) lines to six HrasV12;p53 − null MEF lines, as previously described 3 . Three independent HrasV12;p53 25,26,53,54/25,26,53,54 MEFs lines were analyzed, and showed that the gene expression profiles were indistinguishable from HrasV12;p53 null cells. Numbered columns indicate independent MEF lines. Blue – repressed genes; Red – induced genes. (d) qRT-PCR analysis of p53 target gene expression in untreated MEFs derived from p53 +/+ and p53 25,26,53,54/+ E13.5 embryos. Graphs indicate averages from four independent MEF lines, +/−SD, after normalization to β-actin . **,*** denote p-values of

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Activity Assay, Western Blot, Immunoprecipitation, Plasmid Preparation, Microarray, Expressing, Quantitative RT-PCR, Derivative Assay

    p53 −/−− Embryos Do Not Exhibit Characteristics of CHARGE Syndrome (a) Whole-mount image of the external ear of E15.5 p53 −/− embryo (right) and control embryo (left) showing normal ear pinna development. (b) Whole mount image of E13.5 p53 −/− embryo (right) and control embryo (left) showing normal retinal development and no evidence of coloboma. (c) Whole-mount image of E15.5 p53 −/− embryo (right) and control embryo (left) with normal lower jaw development. (d) Alizarin Red (bone) and Alcian Blue (cartilage) whole-mount stained E14.5 p53 −/− embryo (right) showing normal long bone formation of the ulna (u), humerus (h), mandible (m), and femur (f) relative to control littermate (left). Bottom: Quantification of bone lengths shown as percent of E14.5 littermate controls (n=3). (e) Representative images of H E-stained sagittal sections of E13.5 control (left) and p53 −/− hearts (right) showing all three cardiac cell types in both genotypes. en: endocardium; ep: epicardium; myo: myocardium (arrows). (f) Analysis of H E-stained transverse sections of E13.5 p53 −/− and control hearts revealing normal outflow tract development. (Top) The main pulmonary artery (MPA) and aorta (Ao) are fully septated, and the MPA connects to the right ventricle (RV) in p53 −/− hearts. (Bottom) The aorta connects to the left ventricle (LV). Φ ventricular outflow tract that connects the left ventricle and aorta. PV: pulmonary valve, AV: aortic valve (g) Analysis of transverse sections of H E-stained E13.5 p53 −/− hearts (right) reveals normal atrioventricular cushions which have remodeled to form mature, elongated mitral (mv, arrowhead) and tricuspid (tv, arrow) valves similar to control hearts (left). RA: right atrium; LA: left atrium; RV: right ventricle; LV: left ventricle. (h) H E-stained sagittal section of kidney from p53 −/− (right) and control embryos (left) showing normal renal size and development. (i) H E-stained transverse section of thymi in p53 −/− E13.5 embryo (right) reveals similar thymus size compared to control littermate (left).

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 −/−− Embryos Do Not Exhibit Characteristics of CHARGE Syndrome (a) Whole-mount image of the external ear of E15.5 p53 −/− embryo (right) and control embryo (left) showing normal ear pinna development. (b) Whole mount image of E13.5 p53 −/− embryo (right) and control embryo (left) showing normal retinal development and no evidence of coloboma. (c) Whole-mount image of E15.5 p53 −/− embryo (right) and control embryo (left) with normal lower jaw development. (d) Alizarin Red (bone) and Alcian Blue (cartilage) whole-mount stained E14.5 p53 −/− embryo (right) showing normal long bone formation of the ulna (u), humerus (h), mandible (m), and femur (f) relative to control littermate (left). Bottom: Quantification of bone lengths shown as percent of E14.5 littermate controls (n=3). (e) Representative images of H E-stained sagittal sections of E13.5 control (left) and p53 −/− hearts (right) showing all three cardiac cell types in both genotypes. en: endocardium; ep: epicardium; myo: myocardium (arrows). (f) Analysis of H E-stained transverse sections of E13.5 p53 −/− and control hearts revealing normal outflow tract development. (Top) The main pulmonary artery (MPA) and aorta (Ao) are fully septated, and the MPA connects to the right ventricle (RV) in p53 −/− hearts. (Bottom) The aorta connects to the left ventricle (LV). Φ ventricular outflow tract that connects the left ventricle and aorta. PV: pulmonary valve, AV: aortic valve (g) Analysis of transverse sections of H E-stained E13.5 p53 −/− hearts (right) reveals normal atrioventricular cushions which have remodeled to form mature, elongated mitral (mv, arrowhead) and tricuspid (tv, arrow) valves similar to control hearts (left). RA: right atrium; LA: left atrium; RV: right ventricle; LV: left ventricle. (h) H E-stained sagittal section of kidney from p53 −/− (right) and control embryos (left) showing normal renal size and development. (i) H E-stained transverse section of thymi in p53 −/− E13.5 embryo (right) reveals similar thymus size compared to control littermate (left).

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Staining

    p53 25,26,53,54/+ Embryos Exhibit Lethality and Diverse Craniofacial Defects Characteristic of CHARGE Syndrome (a) p53 TAD mutant allele with L25Q, W26S, F53Q, F54S mutations. Cre deletes Lox-Stop-Lox (LSL) cassette, inducing p53 25,26,53,54 expression. (b) Exencephaly (63%, n=35; arrow) and short lower jaw (74%, n=27; arrowhead) in E15.5 p53 25,26,53,54/+ embryo. (c) Cleft palate (arrow) in E15.5 p53 25,26,53,54/+ embryo. (n=3). (d) Absent external ear pinna (arrow) of E15.5 p53 25,26,53,54/+ embryo. (47%, n=17). (e) Posterior semicircular canal (pc) fused to common crus (CC; arrow) in E13.5 p53 25,26,53,54/+ inner ear. ac-anterior canal, lc-lateral canal. (71%, n=12). ( f) Coloboma (arrow) in E13.5 p53 25,26,53,54/+ embryo. (59%, n=17). (g) Retinal coloboma (Re; arrow) in E15.5 p53 25,26,53,54/+ embryo.

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54/+ Embryos Exhibit Lethality and Diverse Craniofacial Defects Characteristic of CHARGE Syndrome (a) p53 TAD mutant allele with L25Q, W26S, F53Q, F54S mutations. Cre deletes Lox-Stop-Lox (LSL) cassette, inducing p53 25,26,53,54 expression. (b) Exencephaly (63%, n=35; arrow) and short lower jaw (74%, n=27; arrowhead) in E15.5 p53 25,26,53,54/+ embryo. (c) Cleft palate (arrow) in E15.5 p53 25,26,53,54/+ embryo. (n=3). (d) Absent external ear pinna (arrow) of E15.5 p53 25,26,53,54/+ embryo. (47%, n=17). (e) Posterior semicircular canal (pc) fused to common crus (CC; arrow) in E13.5 p53 25,26,53,54/+ inner ear. ac-anterior canal, lc-lateral canal. (71%, n=12). ( f) Coloboma (arrow) in E13.5 p53 25,26,53,54/+ embryo. (59%, n=17). (g) Retinal coloboma (Re; arrow) in E15.5 p53 25,26,53,54/+ embryo.

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Expressing

    p53 25,26,53,54/+ Embryo Tissues Display Increased Apoptosis and Decreased Proliferation (a) Left: Immunofluorescence for Phospho-Histone H3 (red) in the retina of E13.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of Phospho-Histone H3 positive cells per retina area relative to littermate controls. **p-value=0.006 by one-tailed Welsh’s t-test (n=4). ( b ) Left: Immunohistochemistry for cleaved-caspase 3 (CC3) in thymi of control (left) and p53 25,26,53,54/+ (right) embryos. Inset: close-up image of cleaved-caspase 3 positive region. Right: Quantification of CC3-positive cells per thymic area. *p-value=0.02 by one-tailed Student’s t-test (n=4). (c) Immunofluorescence for Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos was used to identify neural crest cells in Figure 2f . (d) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) and Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos. p53 25,26,53,54/+ embryos have more apoptotic (red) neural crest cells, as determined by Pax3-positive staining (green), compared to control littermates. Right: Quantification of CC3 positive cells per total neural crest cell number. p-value=0.14 by one-tailed Student’s t-test (n=4). (e) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) in otic vesicle of E9.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of CC3 positive cells per total cell number. *p-value=0.03 by one-tailed Student’s t-test (n=3). ( f ) Whole-mount cleaved-caspase 3 staining in E8.5 control and p53 25,26,53,54/+ embryos reveals enhanced apoptosis in the neuroepithelium of p53 25,26,53,54/+ embryos (right) but not in controls (left). Close-up shows magnification of the caudal neuroepithelium (bottom). Arrows indicate cleaved-caspase 3 positive regions.

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54/+ Embryo Tissues Display Increased Apoptosis and Decreased Proliferation (a) Left: Immunofluorescence for Phospho-Histone H3 (red) in the retina of E13.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of Phospho-Histone H3 positive cells per retina area relative to littermate controls. **p-value=0.006 by one-tailed Welsh’s t-test (n=4). ( b ) Left: Immunohistochemistry for cleaved-caspase 3 (CC3) in thymi of control (left) and p53 25,26,53,54/+ (right) embryos. Inset: close-up image of cleaved-caspase 3 positive region. Right: Quantification of CC3-positive cells per thymic area. *p-value=0.02 by one-tailed Student’s t-test (n=4). (c) Immunofluorescence for Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos was used to identify neural crest cells in Figure 2f . (d) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) and Pax3 (green) in neural crest cells of E9.5 control and p53 25,26,53,54/+ embryos. p53 25,26,53,54/+ embryos have more apoptotic (red) neural crest cells, as determined by Pax3-positive staining (green), compared to control littermates. Right: Quantification of CC3 positive cells per total neural crest cell number. p-value=0.14 by one-tailed Student’s t-test (n=4). (e) Left: Immunofluorescence for cleaved-caspase 3 (CC3, red) in otic vesicle of E9.5 control and p53 25,26,53,54/+ embryos. Right: Quantification of CC3 positive cells per total cell number. *p-value=0.03 by one-tailed Student’s t-test (n=3). ( f ) Whole-mount cleaved-caspase 3 staining in E8.5 control and p53 25,26,53,54/+ embryos reveals enhanced apoptosis in the neuroepithelium of p53 25,26,53,54/+ embryos (right) but not in controls (left). Close-up shows magnification of the caudal neuroepithelium (bottom). Arrows indicate cleaved-caspase 3 positive regions.

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Immunofluorescence, One-tailed Test, Immunohistochemistry, Staining

    p53 25,26,53,54/+ Embryos Exhibit Additional Features of CHARGE Syndrome (a) H E-stained sections of E12.5 control (left) and p53 25,26,53,54/+ embryos (right). Examination confirmed neural tube closure defects (arrow). (b) Close-up image of UV-illuminated, ethidium bromide-stained E15.5 p53 25,26,53,54/+ embryo (right) to highlight short lower jaw phenotype with protruding tongue (arrow) compared to control littermate (left). 74% (n=27) of p53 25,26,53,54/+ embryos exhibited short lower jaw. Cleft lip not shown. (c) Top: Alizarin Red (bone) and Alcian Blue (cartilage) whole-mount stained E15.0 p53 25,26,53,54/+ embryo (right) showing reduced bone density in the cranium (c) and nasal cavity (n); shorter ulna (u), humerus (h), mandible (m), and femur (f); and reduced bone formation in the ribs (R), where fewer vertebrae are undergoing ossification relative to control littermate (left). Number of vertebrae with bone formation: 19 in control (arrow; V19) versus 18 in p53 25,26,53,54/+ embryo (arrow; V18). The severity of bone and cartilage defects is variable, with the most severe defects evident in embryos with exencephaly and severe craniofacial defects. n=7. Bottom: Quantification of bone lengths shown as percent of E14.5-15.0 littermate controls. Bone lengths of the mandible, humerus, ulna, and femur were measured using the ruler function in Adobe Photoshop on images taken at 6.3×. Only litters with detectable bone formation in p53 25,26,53,54/+ embryos were included in bone length analyses. Student’s T-test **p=0.008 (mandible), **p=0.005 (humerus). (d) Representative images of H E-stained sagittal sections of E12.5 control (left) and p53 25,26,53,54/+ hearts (right) showing all three cardiac cell types in both genotypes. en: endocardium; ep: epicardium; myo: myocardium (arrows). (e) H E-stained E12.5 p53 25,26,53,54/+ heart exhibiting persistent truncus arteriosus (PTA) (33%, n=6). The cardiac outflow tract in the control embryo (left) is septated into the aorta (Ao) and main pulmonary artery (MPA), whereas the cardiac outflow tract (truncus arteriosus or TA) in the p53 25,26,53,54/+ embryo (right) remains unseptated, resulting in PTA. (f) Illustration of control heart (left) and p53 25,26,53,54/+ embryo heart (right), highlighting DORV and atrioventricular cushion defects. Both the aorta (Ao) and main pulmonary artery (MPA) flow out from the right ventricle (RV), resulting in mixed oxygenated and deoxygenated blood in systemic circulation when combined with concurrent VSDs (ventricular septal defects). The atrioventricular cushions remain bulbous and fail to elongate into mature valve leaflets (mitral valve: mv; tricuspid valve: tv). Red: oxygenated blood; Blue: deoxygenated blood; Purple/Pink: mixed oxygenated/deoxygenated blood. (g) Representative H E-stained transverse section of thymus in p53 25,26,53,54/+ E15.5 embryo (right) reveals smaller thymus compared to control littermate (left) (63% of control; n=4). (h) Representative H E analysis of liver sections from E12.5 control (left) and p53 25,26,53,54/+ embryos (right) showing normal liver architecture in both genotypes (top). High magnification image (bottom) of the region of the liver outlined by the white box in the top panel shows the presence of nucleated erythrocytes (arrows), indicating proper hematopoiesis. (i) Top: Table summarizing the incidence (%) and sample size (n) for phenotypes assessed qualitatively in p53 25,26,53,54/+ embryos. The occurrence of these phenotypes in CHARGE syndrome is also indicated (+ present, − absent). Bottom: Table summarizing phenotypes assessed quantitatively in p53 25,26,53,54/+ embryos relative to controls, shown as the percent average size of controls (%), with sample size (n) also indicated. The occurrence of these phenotypes in CHARGE syndrome is also shown (+ present). Detailed description of bone and cartilage defects can be found in Extended-Data Fig. 4c .

    Journal: Nature

    Article Title: Inappropriate p53 Activation During Development Induces Features of CHARGE Syndrome

    doi: 10.1038/nature13585

    Figure Lengend Snippet: p53 25,26,53,54/+ Embryos Exhibit Additional Features of CHARGE Syndrome (a) H E-stained sections of E12.5 control (left) and p53 25,26,53,54/+ embryos (right). Examination confirmed neural tube closure defects (arrow). (b) Close-up image of UV-illuminated, ethidium bromide-stained E15.5 p53 25,26,53,54/+ embryo (right) to highlight short lower jaw phenotype with protruding tongue (arrow) compared to control littermate (left). 74% (n=27) of p53 25,26,53,54/+ embryos exhibited short lower jaw. Cleft lip not shown. (c) Top: Alizarin Red (bone) and Alcian Blue (cartilage) whole-mount stained E15.0 p53 25,26,53,54/+ embryo (right) showing reduced bone density in the cranium (c) and nasal cavity (n); shorter ulna (u), humerus (h), mandible (m), and femur (f); and reduced bone formation in the ribs (R), where fewer vertebrae are undergoing ossification relative to control littermate (left). Number of vertebrae with bone formation: 19 in control (arrow; V19) versus 18 in p53 25,26,53,54/+ embryo (arrow; V18). The severity of bone and cartilage defects is variable, with the most severe defects evident in embryos with exencephaly and severe craniofacial defects. n=7. Bottom: Quantification of bone lengths shown as percent of E14.5-15.0 littermate controls. Bone lengths of the mandible, humerus, ulna, and femur were measured using the ruler function in Adobe Photoshop on images taken at 6.3×. Only litters with detectable bone formation in p53 25,26,53,54/+ embryos were included in bone length analyses. Student’s T-test **p=0.008 (mandible), **p=0.005 (humerus). (d) Representative images of H E-stained sagittal sections of E12.5 control (left) and p53 25,26,53,54/+ hearts (right) showing all three cardiac cell types in both genotypes. en: endocardium; ep: epicardium; myo: myocardium (arrows). (e) H E-stained E12.5 p53 25,26,53,54/+ heart exhibiting persistent truncus arteriosus (PTA) (33%, n=6). The cardiac outflow tract in the control embryo (left) is septated into the aorta (Ao) and main pulmonary artery (MPA), whereas the cardiac outflow tract (truncus arteriosus or TA) in the p53 25,26,53,54/+ embryo (right) remains unseptated, resulting in PTA. (f) Illustration of control heart (left) and p53 25,26,53,54/+ embryo heart (right), highlighting DORV and atrioventricular cushion defects. Both the aorta (Ao) and main pulmonary artery (MPA) flow out from the right ventricle (RV), resulting in mixed oxygenated and deoxygenated blood in systemic circulation when combined with concurrent VSDs (ventricular septal defects). The atrioventricular cushions remain bulbous and fail to elongate into mature valve leaflets (mitral valve: mv; tricuspid valve: tv). Red: oxygenated blood; Blue: deoxygenated blood; Purple/Pink: mixed oxygenated/deoxygenated blood. (g) Representative H E-stained transverse section of thymus in p53 25,26,53,54/+ E15.5 embryo (right) reveals smaller thymus compared to control littermate (left) (63% of control; n=4). (h) Representative H E analysis of liver sections from E12.5 control (left) and p53 25,26,53,54/+ embryos (right) showing normal liver architecture in both genotypes (top). High magnification image (bottom) of the region of the liver outlined by the white box in the top panel shows the presence of nucleated erythrocytes (arrows), indicating proper hematopoiesis. (i) Top: Table summarizing the incidence (%) and sample size (n) for phenotypes assessed qualitatively in p53 25,26,53,54/+ embryos. The occurrence of these phenotypes in CHARGE syndrome is also indicated (+ present, − absent). Bottom: Table summarizing phenotypes assessed quantitatively in p53 25,26,53,54/+ embryos relative to controls, shown as the percent average size of controls (%), with sample size (n) also indicated. The occurrence of these phenotypes in CHARGE syndrome is also shown (+ present). Detailed description of bone and cartilage defects can be found in Extended-Data Fig. 4c .

    Article Snippet: Western blots were probed with anti-p53 (DO-1, Santa Cruz Biotechnology).

    Techniques: Staining, Flow Cytometry

    Seeding effect of P8 fibrils (residues 250 to 257; PILTIITL) on functional p53. (A) Schematic representation of the loss of function of native p53 upon interaction with preformed P8 fibrils (Seed) (i), which leads to change in colony color (ii and iii) and a growth defect (iv), which is due to inactivation of transcription of three reporters, as indicated. (B) Growth curves of strain SGY6003 with (+) and without (−) seed were not found to be significantly different. (C) Internalization of FITC-labeled aggregated P8 fibrils and P8 monomer within the yeast cell. (i) FITC-labeled P8 fibril (green fluorescence) was used to transform yeast cells, similar to what was performed in panels A and E, and we obtained visual confirmation of the internalization of P8 fibrils and their localization in the cytoplasm. (Bottom left) Magnified image of the labeled seed (arrow) inside the cytoplasm. Scale bar, ∼2 μm. (ii) FITC-labeled P8 monomer (green fluorescence) was used to transform yeast cells, similar to what was performed in panels A and E, and we obtained visual confirmation of the internalization of P8 monomer and its localization in the cytoplasm (arrow). Scale bar, ∼2 μm. (D) Immunoprecipitation of p53 from cells with and without seed was performed using anti-p53 antibody. Dot blot analysis was performed with immunoprecipitated p53 using anti-p53, OC, and A11 antibodies separately. WCE, whole-cell extract; p53 IP, immunoprecipitated p53. (E) Immunofluorescence study showing colocalization of p53 and OC antibody in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds, which showed robust colocalized signal with OC antibody. Scale bar, ∼5 μm.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Seeding effect of P8 fibrils (residues 250 to 257; PILTIITL) on functional p53. (A) Schematic representation of the loss of function of native p53 upon interaction with preformed P8 fibrils (Seed) (i), which leads to change in colony color (ii and iii) and a growth defect (iv), which is due to inactivation of transcription of three reporters, as indicated. (B) Growth curves of strain SGY6003 with (+) and without (−) seed were not found to be significantly different. (C) Internalization of FITC-labeled aggregated P8 fibrils and P8 monomer within the yeast cell. (i) FITC-labeled P8 fibril (green fluorescence) was used to transform yeast cells, similar to what was performed in panels A and E, and we obtained visual confirmation of the internalization of P8 fibrils and their localization in the cytoplasm. (Bottom left) Magnified image of the labeled seed (arrow) inside the cytoplasm. Scale bar, ∼2 μm. (ii) FITC-labeled P8 monomer (green fluorescence) was used to transform yeast cells, similar to what was performed in panels A and E, and we obtained visual confirmation of the internalization of P8 monomer and its localization in the cytoplasm (arrow). Scale bar, ∼2 μm. (D) Immunoprecipitation of p53 from cells with and without seed was performed using anti-p53 antibody. Dot blot analysis was performed with immunoprecipitated p53 using anti-p53, OC, and A11 antibodies separately. WCE, whole-cell extract; p53 IP, immunoprecipitated p53. (E) Immunofluorescence study showing colocalization of p53 and OC antibody in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds, which showed robust colocalized signal with OC antibody. Scale bar, ∼5 μm.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Functional Assay, Labeling, Fluorescence, Immunoprecipitation, Dot Blot, Immunofluorescence

    Transmission of aggregated p53 requires the presence of functional p53. Strain SGY6003(pGALp53 LacZ ) was allowed to pass through generations, as indicated, in inducing galactose (Gal) or noninducing dextrose (Dex) medium in the presence or absence of the seeds (P8) or p53 core domain. Finally, the cells were diluted and plated on the X-Gal galactose plate to visualize the colony color. Raf., raffinose.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Transmission of aggregated p53 requires the presence of functional p53. Strain SGY6003(pGALp53 LacZ ) was allowed to pass through generations, as indicated, in inducing galactose (Gal) or noninducing dextrose (Dex) medium in the presence or absence of the seeds (P8) or p53 core domain. Finally, the cells were diluted and plated on the X-Gal galactose plate to visualize the colony color. Raf., raffinose.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Transmission Assay, Functional Assay

    Isolation of p53 amyloids from yeast cell lysate. (A) Amyloid-containing fractions were isolated from yeast cell lysates by sedimentation in SDS-containing lysis buffer. The p53 protein was detected in the total lysate, the SDS-soluble supernatant, and the SDS-insoluble pellet fraction by immunoblotting with anti-p53 antibody. A prestained molecular mass ladder was loaded in the middle lane. The blot was developed with the colorimetric substrate TMB (Genei, India). The blot was developed with anti-GPD to assess GPD levels in fractions with and without seeds as a loading control. (B) Lysates of yeast cells with/without seeds were analyzed by SDD-AGE and Western blotting. The p53 expression was detected by immunoblotting with anti-p53 antibody. Cell lysate from plus-seed cells showing higher-order SDS-resistant aggregates are marked in the gel.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Isolation of p53 amyloids from yeast cell lysate. (A) Amyloid-containing fractions were isolated from yeast cell lysates by sedimentation in SDS-containing lysis buffer. The p53 protein was detected in the total lysate, the SDS-soluble supernatant, and the SDS-insoluble pellet fraction by immunoblotting with anti-p53 antibody. A prestained molecular mass ladder was loaded in the middle lane. The blot was developed with the colorimetric substrate TMB (Genei, India). The blot was developed with anti-GPD to assess GPD levels in fractions with and without seeds as a loading control. (B) Lysates of yeast cells with/without seeds were analyzed by SDD-AGE and Western blotting. The p53 expression was detected by immunoblotting with anti-p53 antibody. Cell lysate from plus-seed cells showing higher-order SDS-resistant aggregates are marked in the gel.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Isolation, Sedimentation, Lysis, Western Blot, Expressing

    Loss of function of aggregated p53. (A) The level of LacZ mRNA was measured in cells with or without seeds using reverse transcription followed by real-time PCR analysis. Two independent reference genes, CDC19 and TAF10 , were used for normalization and fold change in the mRNA levels. The data represent the averages of three independent real-time PCR analyses. The error bars indicate standard deviations from the mean. (B) (Top) ChIP of p53 from cells with or without seeds. Yeast cells were analyzed by PCR and visualized by agarose gel electrophoresis. Differences in amplification were observed in input, IP, and mock IP. TAF10 was used as a nonbinding control. (Bottom) Graphical representation of chromatin immunoprecipitation of p53 displaying percent enrichment/input by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent averages of the results of three independent real-time analyses. (C) Immunofluorescence study of chromatin spread of yeast cells with or without seeds. The cells without seeds, but not those with seeds, displayed p53 in a majority of the spread as chromatin bound. Scale bar, ∼3 μm. Statistical analysis of the total percent spread was calculated as represented graphically below.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Loss of function of aggregated p53. (A) The level of LacZ mRNA was measured in cells with or without seeds using reverse transcription followed by real-time PCR analysis. Two independent reference genes, CDC19 and TAF10 , were used for normalization and fold change in the mRNA levels. The data represent the averages of three independent real-time PCR analyses. The error bars indicate standard deviations from the mean. (B) (Top) ChIP of p53 from cells with or without seeds. Yeast cells were analyzed by PCR and visualized by agarose gel electrophoresis. Differences in amplification were observed in input, IP, and mock IP. TAF10 was used as a nonbinding control. (Bottom) Graphical representation of chromatin immunoprecipitation of p53 displaying percent enrichment/input by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent averages of the results of three independent real-time analyses. (C) Immunofluorescence study of chromatin spread of yeast cells with or without seeds. The cells without seeds, but not those with seeds, displayed p53 in a majority of the spread as chromatin bound. Scale bar, ∼3 μm. Statistical analysis of the total percent spread was calculated as represented graphically below.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Immunofluorescence

    Prion-like infectivity of p53 amyloids. Haploid recipient and donor cells of the indicated genotypes were mated, where donor cells were with (+Seed) or without (−Seed) seeds. Following heterokaryon formation, the cytoductants carrying recipient nuclei and a mixture of donor and recipient cytoplasm were selected based on the recipient nuclear genotype and growth on glycerol medium. The resultant cytoductants were transferred onto a YPD plate to assay ADE2 reporter (A) and onto a His–3-AT plate to assay HIS3 reporter (B).

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Prion-like infectivity of p53 amyloids. Haploid recipient and donor cells of the indicated genotypes were mated, where donor cells were with (+Seed) or without (−Seed) seeds. Following heterokaryon formation, the cytoductants carrying recipient nuclei and a mixture of donor and recipient cytoplasm were selected based on the recipient nuclear genotype and growth on glycerol medium. The resultant cytoductants were transferred onto a YPD plate to assay ADE2 reporter (A) and onto a His–3-AT plate to assay HIS3 reporter (B).

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Infection

    Effect of p53 expression on yeast growth. (A) Growth curves of the yeast strain with and without GAL -p53 vector and with and without seeds on liquid minimal medium in the absence (left) or presence (right) of galactose. The A 600 was measured every 2 h. The data points represent the means of the results of three independent experiments. The error bars indicate standard deviations from the mean. (B) Analysis of cell death induced in yeast. Addition of the seeds drastically reduced the percentage of cells stained with the death markers annexin V and PI. Scale bar, ∼5 μm.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Effect of p53 expression on yeast growth. (A) Growth curves of the yeast strain with and without GAL -p53 vector and with and without seeds on liquid minimal medium in the absence (left) or presence (right) of galactose. The A 600 was measured every 2 h. The data points represent the means of the results of three independent experiments. The error bars indicate standard deviations from the mean. (B) Analysis of cell death induced in yeast. Addition of the seeds drastically reduced the percentage of cells stained with the death markers annexin V and PI. Scale bar, ∼5 μm.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Expressing, Plasmid Preparation, Staining

    Rescue of p53 amyloid due to overexpression of Hsp104. (A) Live-cell imaging to visualize p53-GFP signal in cells overexpressing Hsp104. Three types of cells were observed: type I, where cytoplasmic signal of p53-GFP was observed, suggesting localization of p53 aggregates in the cytoplasm; type II, where nuclear signal of p53-GFP was observed, suggesting functional nonaggregating p53 enters the nucleus; and type III, where both cytoplasmic and nuclear signals were observed, suggesting a fraction of p53 aggregates were rescued and became functional due to Hsp104 overexpression. Scale bar, 5 μm. (B) Field view showing all three types of cells. Scale bar, 3 μm. (C) Percentage distributions of the three types of cells with or without Hsp104. (D) Dose-dependent curing of p53 prion by Hsp104. (E) Growth defect observed due to inactivation of transcription of the HIS3 reporter for the cells harboring seeds. However, the defect was partially rescued when the same cells overexpressed Hsp104 (compare the rows indicated by the red and green arrowheads). (F) The growth rescue effect of Hsp104 in panel E was abolished upon treatment with GdHCl, and the growth of the treated cells became similar to the that of the plus-seed cells (compare the rows indicated by the red arrowheads).

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Rescue of p53 amyloid due to overexpression of Hsp104. (A) Live-cell imaging to visualize p53-GFP signal in cells overexpressing Hsp104. Three types of cells were observed: type I, where cytoplasmic signal of p53-GFP was observed, suggesting localization of p53 aggregates in the cytoplasm; type II, where nuclear signal of p53-GFP was observed, suggesting functional nonaggregating p53 enters the nucleus; and type III, where both cytoplasmic and nuclear signals were observed, suggesting a fraction of p53 aggregates were rescued and became functional due to Hsp104 overexpression. Scale bar, 5 μm. (B) Field view showing all three types of cells. Scale bar, 3 μm. (C) Percentage distributions of the three types of cells with or without Hsp104. (D) Dose-dependent curing of p53 prion by Hsp104. (E) Growth defect observed due to inactivation of transcription of the HIS3 reporter for the cells harboring seeds. However, the defect was partially rescued when the same cells overexpressed Hsp104 (compare the rows indicated by the red and green arrowheads). (F) The growth rescue effect of Hsp104 in panel E was abolished upon treatment with GdHCl, and the growth of the treated cells became similar to the that of the plus-seed cells (compare the rows indicated by the red arrowheads).

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Over Expression, Live Cell Imaging, Functional Assay

    Effect of P8 fibril on GFP-tagged functional p53. (A) Immunofluorescence assay to visualize p53-GFP in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds. However, in cells without any seeds, p53-GFP was colocalized with DAPI. Scale bar, ∼5 μm. (B) Graphical representation of ChIP of p53 displaying percent enrichment/input at the p53 binding element by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent the averages of the results of three independent real-time analyses. The error bars indicate standard deviations from the mean.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Effect of P8 fibril on GFP-tagged functional p53. (A) Immunofluorescence assay to visualize p53-GFP in yeast cells with or without seeds. Aggregates of p53 are seen as green cytoplasmic focus structures specifically in the cells harboring the seeds. However, in cells without any seeds, p53-GFP was colocalized with DAPI. Scale bar, ∼5 μm. (B) Graphical representation of ChIP of p53 displaying percent enrichment/input at the p53 binding element by real-time qPCR analysis. The antibody used for ChIP was anti-p53 D0-1 (5 μg). The data represent the averages of the results of three independent real-time analyses. The error bars indicate standard deviations from the mean.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Functional Assay, Immunofluorescence, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction

    Aggregation of native p53 requires preaggregated amyloidogenic peptides or the core domain. Loss of function of native p53 in the strain SGY6003(pGALp53 LacZ ) occurs in the presence of preaggregated amyloidogenic fibrils (PILTIITL) (A) or the core domain (B). However, monomeric amyloidogenic peptide, core domain, or aggregated scrambled peptide (ITLPITLI) failed to inactivate the native p53 function.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Aggregation of native p53 requires preaggregated amyloidogenic peptides or the core domain. Loss of function of native p53 in the strain SGY6003(pGALp53 LacZ ) occurs in the presence of preaggregated amyloidogenic fibrils (PILTIITL) (A) or the core domain (B). However, monomeric amyloidogenic peptide, core domain, or aggregated scrambled peptide (ITLPITLI) failed to inactivate the native p53 function.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques:

    Dominant character and non-Mendelian segregation by p53 amyloids. (A) Haploid pGALp53 LacZ (+ Seed) and pADHp53 ADE2 (− Seed) haploids were streaked on a YPD plate. The two strains were mated to obtain the desired combination. The resultant strain was streaked on YPD to check the colony assay, which was observed to be plus-seed cells, suggesting the presence of a dominant trait. (B) (Right) p53 amyloids in a diploid cell obtained by mating between the plus-seed and the minus-seed cells displayed non-Mendelian segregation, since all the meiotic spores of almost all the dissected tetrads displayed nonfunctional and aggregated p53, as shown by white colonies. (Left) Mating between the minus-seed cells (control) showing all the meiotic spores carrying functional p53, as shown by blue colonies.

    Journal: Molecular and Cellular Biology

    Article Title: Evidence of a Prion-Like Transmission of p53 Amyloid in Saccharomyces cerevisiae

    doi: 10.1128/MCB.00118-17

    Figure Lengend Snippet: Dominant character and non-Mendelian segregation by p53 amyloids. (A) Haploid pGALp53 LacZ (+ Seed) and pADHp53 ADE2 (− Seed) haploids were streaked on a YPD plate. The two strains were mated to obtain the desired combination. The resultant strain was streaked on YPD to check the colony assay, which was observed to be plus-seed cells, suggesting the presence of a dominant trait. (B) (Right) p53 amyloids in a diploid cell obtained by mating between the plus-seed and the minus-seed cells displayed non-Mendelian segregation, since all the meiotic spores of almost all the dissected tetrads displayed nonfunctional and aggregated p53, as shown by white colonies. (Left) Mating between the minus-seed cells (control) showing all the meiotic spores carrying functional p53, as shown by blue colonies.

    Article Snippet: Immunofluorescence assays were performed as described previously ( ) using anti-p53 antibody (1:1,000; Santa Cruz Biotechnology) and secondary antibody (tetramethyl rhodamine [TRITC]-conjugated anti-goat mouse antibody, 1:200 dilution; Jackson, USA).

    Techniques: Colony Assay, Functional Assay

    (A) Lentiviral vector construct expressing Kras G12D mutation under the control of the cytomegalovirus promoter. We incorporated into our lentiviral vector construct a woodchuck hepatitis virus post-transcriptional regulatory element at the 3’ untranslated region of coding sequence; this substantially increased the level of expression of the transgene. (B) The shRNA p53 was introduced into a lentivirus construct under the control of U6 promoter. Both constructs co-express the luciferase gene. (C) Western blots of pancreatic tissues 30 weeks post-injection with lentivirus shRNA p53 and Lentivirus Kras G12D and non-cancer control mice. Non-pancreatic cancer mouse model (control) was injected with lentivirus shRNA-scramble and with lentivirus-expressing GFP at the same concentration and volume as the pancreatic cancer mouse model.

    Journal: Carcinogenesis

    Article Title: A novel experimental model for human mixed acinar–ductal pancreatic cancer

    doi: 10.1093/carcin/bgx119

    Figure Lengend Snippet: (A) Lentiviral vector construct expressing Kras G12D mutation under the control of the cytomegalovirus promoter. We incorporated into our lentiviral vector construct a woodchuck hepatitis virus post-transcriptional regulatory element at the 3’ untranslated region of coding sequence; this substantially increased the level of expression of the transgene. (B) The shRNA p53 was introduced into a lentivirus construct under the control of U6 promoter. Both constructs co-express the luciferase gene. (C) Western blots of pancreatic tissues 30 weeks post-injection with lentivirus shRNA p53 and Lentivirus Kras G12D and non-cancer control mice. Non-pancreatic cancer mouse model (control) was injected with lentivirus shRNA-scramble and with lentivirus-expressing GFP at the same concentration and volume as the pancreatic cancer mouse model.

    Article Snippet: The western blot of pancreatic cancer tissue from adults wild-type mice injected with lentivirus—shRNA p53 and lentivirus—KrasG12D directly to the pancreas was carried out using the anti-Kras antibody and p53 antibody from Santa Cruz Biotechnology at 1:1000 dilution, followed by a horseradish peroxidase-conjugated secondary antibody.

    Techniques: Plasmid Preparation, Construct, Expressing, Mutagenesis, Sequencing, shRNA, Luciferase, Western Blot, Injection, Mouse Assay, Concentration Assay

    Progression of pancreatic cancer in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (A) Histology of pancreas staining for Ki67 in control mice injected with lentivirus shRNA-scramble and GFP. ( B ) Histology staining of pancreatic cancer for ki67 (brown) in mice injected with lentivirus shRNA p53 and kras G12D . (C) PanIN formation in spontaneous pancreatic cancer in adult wild-type mice injected with lentivirus expressing shRNA p53 and Kras G12D , directly into the pancreas. Histologic section showed the formation of PanIN grade 3. (D) Histology of pancreatic carcinoma in mice injected with shRNA p53 and Kras G12D . (E) Western blot detection of cytokeratin 18 in pancreatic tissues 30 weeks post-injection with lentivirus shRNA p53 and Lentivirus Kras G12D and in non-cancer control mice.

    Journal: Carcinogenesis

    Article Title: A novel experimental model for human mixed acinar–ductal pancreatic cancer

    doi: 10.1093/carcin/bgx119

    Figure Lengend Snippet: Progression of pancreatic cancer in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (A) Histology of pancreas staining for Ki67 in control mice injected with lentivirus shRNA-scramble and GFP. ( B ) Histology staining of pancreatic cancer for ki67 (brown) in mice injected with lentivirus shRNA p53 and kras G12D . (C) PanIN formation in spontaneous pancreatic cancer in adult wild-type mice injected with lentivirus expressing shRNA p53 and Kras G12D , directly into the pancreas. Histologic section showed the formation of PanIN grade 3. (D) Histology of pancreatic carcinoma in mice injected with shRNA p53 and Kras G12D . (E) Western blot detection of cytokeratin 18 in pancreatic tissues 30 weeks post-injection with lentivirus shRNA p53 and Lentivirus Kras G12D and in non-cancer control mice.

    Article Snippet: The western blot of pancreatic cancer tissue from adults wild-type mice injected with lentivirus—shRNA p53 and lentivirus—KrasG12D directly to the pancreas was carried out using the anti-Kras antibody and p53 antibody from Santa Cruz Biotechnology at 1:1000 dilution, followed by a horseradish peroxidase-conjugated secondary antibody.

    Techniques: Mouse Assay, Injection, Expressing, shRNA, Staining, Western Blot

    Progression of pancreatic cancer in adults wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (A) and (B) Activated Erk1/2 pancreatic cancer in adult wild-type mice with lentivirus expressing, shRNA p53 and Kras G12D , injected directly into the pancreas. (A) and (B) Immunohistochemical staining for activated Erk1/2 in the pancreatic tissues section cancer model. The brown color indicates immunoreactivity for activated (phosphorylated) Erk1/2. (C) and (D) Immunohistochemical staining for mucin 4 (brown) in pancreatic tissue section from the mouse cancer model. (E) Immunohistochemical staining for Bcl10 (brown) in the pancreatic tissue section from the mouse cancer model. (F) and (G) Immunohistochemical staining for trypsin (brown) in the pancreatic tissue section from the mouse cancer model. (H) Quantitative reverse transcription–polymerase chain reaction demonstrating increased mRNA level of MUC1 in pancreatic tissues from the mouse cancer model compared with control group.

    Journal: Carcinogenesis

    Article Title: A novel experimental model for human mixed acinar–ductal pancreatic cancer

    doi: 10.1093/carcin/bgx119

    Figure Lengend Snippet: Progression of pancreatic cancer in adults wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (A) and (B) Activated Erk1/2 pancreatic cancer in adult wild-type mice with lentivirus expressing, shRNA p53 and Kras G12D , injected directly into the pancreas. (A) and (B) Immunohistochemical staining for activated Erk1/2 in the pancreatic tissues section cancer model. The brown color indicates immunoreactivity for activated (phosphorylated) Erk1/2. (C) and (D) Immunohistochemical staining for mucin 4 (brown) in pancreatic tissue section from the mouse cancer model. (E) Immunohistochemical staining for Bcl10 (brown) in the pancreatic tissue section from the mouse cancer model. (F) and (G) Immunohistochemical staining for trypsin (brown) in the pancreatic tissue section from the mouse cancer model. (H) Quantitative reverse transcription–polymerase chain reaction demonstrating increased mRNA level of MUC1 in pancreatic tissues from the mouse cancer model compared with control group.

    Article Snippet: The western blot of pancreatic cancer tissue from adults wild-type mice injected with lentivirus—shRNA p53 and lentivirus—KrasG12D directly to the pancreas was carried out using the anti-Kras antibody and p53 antibody from Santa Cruz Biotechnology at 1:1000 dilution, followed by a horseradish peroxidase-conjugated secondary antibody.

    Techniques: Mouse Assay, Injection, Expressing, shRNA, Immunohistochemistry, Staining, Reverse Transcription Polymerase Chain Reaction

    (A) Tumor growth monitoring in a cohort of four mice began at 8 weeks of age [time zero] and 1 week before injection of lentivirus oncogenes KrasG12D and shRNA p53 and was followed over 30 weeks by in vivo bioluminescence. The signal intensity was normalized for each mouse individually, with 100% representing the geometrical mean of all values obtained per mouse during the 30 weeks observation period. For quantitation of light intensity in time course experiments, a constant analysis gate was defined and individual tumor photos were determined by centering the gate on the highest signal intensity for each time point. The mean ± SEM of the four mice is shown. (B) and (C) Pancreatic tumor progression in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (B) Pancreatic tissue demonstrated the formation of a tubular complex with surrounding fibroblasts. (C) Periodic acid–Schiff staining is demonstrating mucin accumulation.

    Journal: Carcinogenesis

    Article Title: A novel experimental model for human mixed acinar–ductal pancreatic cancer

    doi: 10.1093/carcin/bgx119

    Figure Lengend Snippet: (A) Tumor growth monitoring in a cohort of four mice began at 8 weeks of age [time zero] and 1 week before injection of lentivirus oncogenes KrasG12D and shRNA p53 and was followed over 30 weeks by in vivo bioluminescence. The signal intensity was normalized for each mouse individually, with 100% representing the geometrical mean of all values obtained per mouse during the 30 weeks observation period. For quantitation of light intensity in time course experiments, a constant analysis gate was defined and individual tumor photos were determined by centering the gate on the highest signal intensity for each time point. The mean ± SEM of the four mice is shown. (B) and (C) Pancreatic tumor progression in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (B) Pancreatic tissue demonstrated the formation of a tubular complex with surrounding fibroblasts. (C) Periodic acid–Schiff staining is demonstrating mucin accumulation.

    Article Snippet: The western blot of pancreatic cancer tissue from adults wild-type mice injected with lentivirus—shRNA p53 and lentivirus—KrasG12D directly to the pancreas was carried out using the anti-Kras antibody and p53 antibody from Santa Cruz Biotechnology at 1:1000 dilution, followed by a horseradish peroxidase-conjugated secondary antibody.

    Techniques: Mouse Assay, Injection, shRNA, In Vivo, Quantitation Assay, Expressing, Staining

    In vivo luciferase bioluminescence of adult wild-type mice injected with lentivirus Kras G12D and lentivirus shRNA p53 directly into the pancreas. BLI of spontaneous pancreatic cancer arising from lentivirus—shRNA p53 and Lentivirus—Kras G12D in vivo . Both lentivirus Kras G12D and shRNA p53 co-express the luciferase gene for tumor detection by Xenogen IVIS system that allows visualization of tumors with bioluminescence in vivo . (A) In vivo imaging demonstrated luminescence signals from pancreatic tumors, which developed following intraductal pancreatic injection of lentivirus Kras G12D and shRNA p53 in wild-type adult mice at weeks 28, 29 and 30. (B) Imaging of internal organs after abdominal incision. Wild-type adult mice showed bioluminescence signals in the pancreas injected with lentivirus Kras G12D and shRNA p53.

    Journal: Carcinogenesis

    Article Title: A novel experimental model for human mixed acinar–ductal pancreatic cancer

    doi: 10.1093/carcin/bgx119

    Figure Lengend Snippet: In vivo luciferase bioluminescence of adult wild-type mice injected with lentivirus Kras G12D and lentivirus shRNA p53 directly into the pancreas. BLI of spontaneous pancreatic cancer arising from lentivirus—shRNA p53 and Lentivirus—Kras G12D in vivo . Both lentivirus Kras G12D and shRNA p53 co-express the luciferase gene for tumor detection by Xenogen IVIS system that allows visualization of tumors with bioluminescence in vivo . (A) In vivo imaging demonstrated luminescence signals from pancreatic tumors, which developed following intraductal pancreatic injection of lentivirus Kras G12D and shRNA p53 in wild-type adult mice at weeks 28, 29 and 30. (B) Imaging of internal organs after abdominal incision. Wild-type adult mice showed bioluminescence signals in the pancreas injected with lentivirus Kras G12D and shRNA p53.

    Article Snippet: The western blot of pancreatic cancer tissue from adults wild-type mice injected with lentivirus—shRNA p53 and lentivirus—KrasG12D directly to the pancreas was carried out using the anti-Kras antibody and p53 antibody from Santa Cruz Biotechnology at 1:1000 dilution, followed by a horseradish peroxidase-conjugated secondary antibody.

    Techniques: In Vivo, Luciferase, Mouse Assay, Injection, shRNA, In Vivo Imaging, Imaging

    Progression of pancreatic cancer in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (A) and (B) Immunohistochemical staining for cytokeratin 7 (brown) in adult wild-type mice with lentivirus expressing, shRNA p53 and Kras G12D , injected directly into the pancreas. (C) and (D) Immunohistochemical staining for cytokeratin 19 (brown) in adult wild-type mice with lentivirus expressing, shRNA p53 and Kras G12D , injected directly into the pancreas. (E) H E stained demonstrated acinar cell carcinoma solid pattern with uniform round nuclei. (F) H E stained of pancreatic carcinoma. (G) H E stained demonstrated developing tubular complex and surrounding fibroblasts. (H) H E stained section showed the formation of PanIN grade 3. H E = Hematoxylin and eosin stain.

    Journal: Carcinogenesis

    Article Title: A novel experimental model for human mixed acinar–ductal pancreatic cancer

    doi: 10.1093/carcin/bgx119

    Figure Lengend Snippet: Progression of pancreatic cancer in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (A) and (B) Immunohistochemical staining for cytokeratin 7 (brown) in adult wild-type mice with lentivirus expressing, shRNA p53 and Kras G12D , injected directly into the pancreas. (C) and (D) Immunohistochemical staining for cytokeratin 19 (brown) in adult wild-type mice with lentivirus expressing, shRNA p53 and Kras G12D , injected directly into the pancreas. (E) H E stained demonstrated acinar cell carcinoma solid pattern with uniform round nuclei. (F) H E stained of pancreatic carcinoma. (G) H E stained demonstrated developing tubular complex and surrounding fibroblasts. (H) H E stained section showed the formation of PanIN grade 3. H E = Hematoxylin and eosin stain.

    Article Snippet: The western blot of pancreatic cancer tissue from adults wild-type mice injected with lentivirus—shRNA p53 and lentivirus—KrasG12D directly to the pancreas was carried out using the anti-Kras antibody and p53 antibody from Santa Cruz Biotechnology at 1:1000 dilution, followed by a horseradish peroxidase-conjugated secondary antibody.

    Techniques: Mouse Assay, Injection, Expressing, shRNA, Immunohistochemistry, Staining, H&E Stain

    (A) Percent (%) of wild-type mice injected with lentivirus-shRNA p53 and lentivirus-Kras G12D directly into the pancreas, which developed primary pancreatic tumors. (B) Percentage of mice with pancreatic cancer marker findings ( n = 7). (C) Gross and microscopic examination of H E stained sections were used to identify the percentage of mice with at least one metastasis to the liver and lung. (D) and (E) Metastatic progression in spontaneous pancreatic cancer in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (D) Liver metastases (arrow). (E) Lung metastases (arrow). (F) and (G) Immunohistochemical staining for Chromagranin A (brown) in the pancreatic tissue section from the mouse cancer model.

    Journal: Carcinogenesis

    Article Title: A novel experimental model for human mixed acinar–ductal pancreatic cancer

    doi: 10.1093/carcin/bgx119

    Figure Lengend Snippet: (A) Percent (%) of wild-type mice injected with lentivirus-shRNA p53 and lentivirus-Kras G12D directly into the pancreas, which developed primary pancreatic tumors. (B) Percentage of mice with pancreatic cancer marker findings ( n = 7). (C) Gross and microscopic examination of H E stained sections were used to identify the percentage of mice with at least one metastasis to the liver and lung. (D) and (E) Metastatic progression in spontaneous pancreatic cancer in adult wild-type mice injected with lentivirus expressing, shRNA p53 and Kras G12D , directly into the pancreas. (D) Liver metastases (arrow). (E) Lung metastases (arrow). (F) and (G) Immunohistochemical staining for Chromagranin A (brown) in the pancreatic tissue section from the mouse cancer model.

    Article Snippet: The western blot of pancreatic cancer tissue from adults wild-type mice injected with lentivirus—shRNA p53 and lentivirus—KrasG12D directly to the pancreas was carried out using the anti-Kras antibody and p53 antibody from Santa Cruz Biotechnology at 1:1000 dilution, followed by a horseradish peroxidase-conjugated secondary antibody.

    Techniques: Mouse Assay, Injection, shRNA, Marker, Staining, Expressing, Immunohistochemistry

    p53 occupancy at genes in developmental pathways in the kidney. p53 ChIP-Seq tracks of select genes of FGF ( A, B ), WNT-β-catenin ( C, D ), BMP ( E, F ), and NOTCH ( G, H ) signaling pathways. Scale bars for peak height are shown. Green arrows indicate

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: p53 occupancy at genes in developmental pathways in the kidney. p53 ChIP-Seq tracks of select genes of FGF ( A, B ), WNT-β-catenin ( C, D ), BMP ( E, F ), and NOTCH ( G, H ) signaling pathways. Scale bars for peak height are shown. Green arrows indicate

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Chromatin Immunoprecipitation

    p53 regulated developmental pathways. p53-mediated gene regulation of genes in FGF, WNT-β-catenin, BMP, and NOTCH signaling pathways (Ingenuity Pathway Analysis) is shown. The schematics illustrate the large number of genes in each pathway that

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: p53 regulated developmental pathways. p53-mediated gene regulation of genes in FGF, WNT-β-catenin, BMP, and NOTCH signaling pathways (Ingenuity Pathway Analysis) is shown. The schematics illustrate the large number of genes in each pathway that

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques:

    Validation of genes altered in microarray of embryonic day (E)15.5 p53-null kidneys. Some known p53 target genes (blue zone), key nephrogenesis regulatory genes (red zone), and other significantly regulated genes (green zone) were validated by QPCR. Significant

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: Validation of genes altered in microarray of embryonic day (E)15.5 p53-null kidneys. Some known p53 target genes (blue zone), key nephrogenesis regulatory genes (red zone), and other significantly regulated genes (green zone) were validated by QPCR. Significant

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Microarray, Real-time Polymerase Chain Reaction

    p53 occupancy at key developmental regulatory genes. Peaks associated with some genes involved in early kidney development visualized as ChIP tracks in Integrated Genome Browser (IGB). Scale bars for peak height are shown. Green arrows indicate TSS and

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: p53 occupancy at key developmental regulatory genes. Peaks associated with some genes involved in early kidney development visualized as ChIP tracks in Integrated Genome Browser (IGB). Scale bars for peak height are shown. Green arrows indicate TSS and

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Chromatin Immunoprecipitation

    Reporter analysis of novel p53-bound genes. PCR-amplified gene promoters containing peak regions were cloned into a promoterless pGL3 basic vector. p53-null H1299 cells were transiently cotransfected with a p53 expression plasmid (pCMV-p53) and the reporter

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: Reporter analysis of novel p53-bound genes. PCR-amplified gene promoters containing peak regions were cloned into a promoterless pGL3 basic vector. p53-null H1299 cells were transiently cotransfected with a p53 expression plasmid (pCMV-p53) and the reporter

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Expressing

    Identification of p53-target genes in the developing kidney.

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: Identification of p53-target genes in the developing kidney.

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques:

    The p53-regulated transcriptome in the developing kidney. A : gene expression analysis in p53−/− E15.5 kidneys. The primary analysis of the original signal readouts reveal a considerable amount of significantly regulated probes (red dots,

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: The p53-regulated transcriptome in the developing kidney. A : gene expression analysis in p53−/− E15.5 kidneys. The primary analysis of the original signal readouts reveal a considerable amount of significantly regulated probes (red dots,

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Expressing

    Identification of p53-target genes in the developing kidney.

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: Identification of p53-target genes in the developing kidney.

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques:

    Identification of p53-target genes in the developing kidney.

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: Identification of p53-target genes in the developing kidney.

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques:

    p53 genome-binding profile in the developing kidney. A : number of unique, mapped reads in p53-chromatin immunoprecipitation (ChIP) and non-ChIP (Input) sample, and model-based analysis of ChIP-Seq (MACS)-identified peaks. B : distribution of p53 intervals

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: p53 genome-binding profile in the developing kidney. A : number of unique, mapped reads in p53-chromatin immunoprecipitation (ChIP) and non-ChIP (Input) sample, and model-based analysis of ChIP-Seq (MACS)-identified peaks. B : distribution of p53 intervals

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Magnetic Cell Separation

    p53-regulated transcriptome in the developing kidney.

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: p53-regulated transcriptome in the developing kidney.

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques:

    p53 enrichment at classical target genes in the developing kidney. p53 binding enrichments are visualized as ChIP-Seq tracks in Integrated Genome Browser. Scale bars for peak height are shown. Green arrows indicate TSS and the direction of transcription.

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: p53 enrichment at classical target genes in the developing kidney. p53 binding enrichments are visualized as ChIP-Seq tracks in Integrated Genome Browser. Scale bars for peak height are shown. Green arrows indicate TSS and the direction of transcription.

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Binding Assay, Chromatin Immunoprecipitation

    Enrichment of p53 targets in various compartments of the developing nephron. A : scheme of a developing nephron. Left : a nascent nephron is produced via cap mesenchyme (CM) induction into renal vesicle (RV) and RV segmentation into comma-shaped (CB) and

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: Enrichment of p53 targets in various compartments of the developing nephron. A : scheme of a developing nephron. Left : a nascent nephron is produced via cap mesenchyme (CM) induction into renal vesicle (RV) and RV segmentation into comma-shaped (CB) and

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Produced

    ChIP tracks of p53 high-occupancy genes viewed in IGB. A : Per1; B : Naif1; C : Cbx3; D : Hspa9; E : Ccdc47. Scale bars for peak height are shown. Green arrows indicate TSS and direction of transcription.

    Journal: Physiological Genomics

    Article Title: Genome-wide analysis of the p53 gene regulatory network in the developing mouse kidney

    doi: 10.1152/physiolgenomics.00113.2013

    Figure Lengend Snippet: ChIP tracks of p53 high-occupancy genes viewed in IGB. A : Per1; B : Naif1; C : Cbx3; D : Hspa9; E : Ccdc47. Scale bars for peak height are shown. Green arrows indicate TSS and direction of transcription.

    Article Snippet: ChIP was performed using an antibody against p53 (Santa Cruz, SC6243X) on chromatin prepared from E15.5 C57BL/6 mice kidneys.

    Techniques: Chromatin Immunoprecipitation

    Ex vivo analysis of tumour slices by two-photon microscopy. ( a ) Ex vivo tumour slice from NICD/p53 −/− mice observed by two-photon microscopy and represented as mosaic of 9 × 9 connected tiles. Epithelial cells have GFP-positive nuclei (green), second harmonic generation (SHG, pink) reveals the presence of collagen I. Insets represent higher magnification of several tumour areas (1–3 tumour bulk; 4–6 invasive areas). Scale bar, 100 μm. ( b – d ) Several examples of ex vivo tumour slices from NICD/p53 −/− mice. Epithelial cells (GFP-positive nuclei, green), collagen I (SHG, pink), all cells (membrane dye, FM 4-64, blue). Images are taken from a Z-stack with 6 μm between planes. ( b ) Invasive front of the tumour. Note that stromal cells surround cancer cells. ( c ) Cancer cells invading stroma as clusters and strings parallel to the collagen fibres. ( d ) Cluster and single cancer cells invading collagen-rich stroma. Single cancer cells are labelled with white arrowheads. Inset, higher magnification of individual cancer cell with elongated nucleus. Scale bar, 100 μm.

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: Ex vivo analysis of tumour slices by two-photon microscopy. ( a ) Ex vivo tumour slice from NICD/p53 −/− mice observed by two-photon microscopy and represented as mosaic of 9 × 9 connected tiles. Epithelial cells have GFP-positive nuclei (green), second harmonic generation (SHG, pink) reveals the presence of collagen I. Insets represent higher magnification of several tumour areas (1–3 tumour bulk; 4–6 invasive areas). Scale bar, 100 μm. ( b – d ) Several examples of ex vivo tumour slices from NICD/p53 −/− mice. Epithelial cells (GFP-positive nuclei, green), collagen I (SHG, pink), all cells (membrane dye, FM 4-64, blue). Images are taken from a Z-stack with 6 μm between planes. ( b ) Invasive front of the tumour. Note that stromal cells surround cancer cells. ( c ) Cancer cells invading stroma as clusters and strings parallel to the collagen fibres. ( d ) Cluster and single cancer cells invading collagen-rich stroma. Single cancer cells are labelled with white arrowheads. Inset, higher magnification of individual cancer cell with elongated nucleus. Scale bar, 100 μm.

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Ex Vivo, Microscopy, Mouse Assay

    Schematic representation of phenotypes observed in single and double mutants. When DNA damage occurs in a healthy cell, the cell can undergo apoptosis if the damage is severe enough. If another mutation then occurs, this can lead to different cell phenotypes depending on the kind of mutation: Apc loss-of-function mutation, as well as Notch activation will give rise to increased proliferation. A p53 loss-of-function mutation will give rise to a cell phenotype that is able to survive, as apoptosis can no longer be induced by p53. A sequential mutation will lead to either adenoma or metastases depending on the preceding mutation. MET: mesenchymal epithelial transition. The predicted phenotypes have been confirmed by experiments.

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: Schematic representation of phenotypes observed in single and double mutants. When DNA damage occurs in a healthy cell, the cell can undergo apoptosis if the damage is severe enough. If another mutation then occurs, this can lead to different cell phenotypes depending on the kind of mutation: Apc loss-of-function mutation, as well as Notch activation will give rise to increased proliferation. A p53 loss-of-function mutation will give rise to a cell phenotype that is able to survive, as apoptosis can no longer be induced by p53. A sequential mutation will lead to either adenoma or metastases depending on the preceding mutation. MET: mesenchymal epithelial transition. The predicted phenotypes have been confirmed by experiments.

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Mutagenesis, Activation Assay

    Characterization of NICD/p53 −/− primary tumours and metastases. ( a ) Kaplan–Meier analysis of NICD/p53 −/− mice and their single transgenic littermates NICD (pink) and p53 −/− (green) over a period of 2 years. The triple transgenic mice (blue) are all dead by 15 months with gut adenocarcinoma, whereas control littermates (pink and green) are dying after 15 months. ( b ) Tumour intake in the intestinal tract of NICD/p53 −/− cohort mice and their relative control littermates at different time points after tamoxifen induction. NICD/p53 −/− mice (blue) develop intestinal adenocarcinoma faster and with a higher penetrance than control littermates (pink and green). ( c ) Macroscopic view of a primary invasive adenocarcinoma located in the jejunum of a NICD/p53 −/− mouse. Scale bar, 1 cm. ( d ) haematoxylin and eosin staining (H E) staining on paraffin-embedded primary tumour from NICD/p53 −/− mice, representing main features of the tumour. Cancer cells are invading all layers of normal tissue to reach the serosa. We suspect that elongated, single cancer cells are present in desmoplastic areas. Scale bar, 40 μm. ( e ) H E and ( f ) nuclear GFP stainings showing primary tumour, lymph node and liver metastases, and peritoneal carcinomatosis in NICD/p53 −/− compound mice. Notice the nonspecific staining due to autofluorescence in the surrounding tumour tissue of lymph node (LN) or liver metastasis and peritoneum carcinomatosis. Scale bars, 40 μm. ( g ) Immunohistochemical staining of β-catenin showing its nuclear delocalization in cancer cells and its overexpression at the tumour front. Scale bars, 40 μm.

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: Characterization of NICD/p53 −/− primary tumours and metastases. ( a ) Kaplan–Meier analysis of NICD/p53 −/− mice and their single transgenic littermates NICD (pink) and p53 −/− (green) over a period of 2 years. The triple transgenic mice (blue) are all dead by 15 months with gut adenocarcinoma, whereas control littermates (pink and green) are dying after 15 months. ( b ) Tumour intake in the intestinal tract of NICD/p53 −/− cohort mice and their relative control littermates at different time points after tamoxifen induction. NICD/p53 −/− mice (blue) develop intestinal adenocarcinoma faster and with a higher penetrance than control littermates (pink and green). ( c ) Macroscopic view of a primary invasive adenocarcinoma located in the jejunum of a NICD/p53 −/− mouse. Scale bar, 1 cm. ( d ) haematoxylin and eosin staining (H E) staining on paraffin-embedded primary tumour from NICD/p53 −/− mice, representing main features of the tumour. Cancer cells are invading all layers of normal tissue to reach the serosa. We suspect that elongated, single cancer cells are present in desmoplastic areas. Scale bar, 40 μm. ( e ) H E and ( f ) nuclear GFP stainings showing primary tumour, lymph node and liver metastases, and peritoneal carcinomatosis in NICD/p53 −/− compound mice. Notice the nonspecific staining due to autofluorescence in the surrounding tumour tissue of lymph node (LN) or liver metastasis and peritoneum carcinomatosis. Scale bars, 40 μm. ( g ) Immunohistochemical staining of β-catenin showing its nuclear delocalization in cancer cells and its overexpression at the tumour front. Scale bars, 40 μm.

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Mouse Assay, Transgenic Assay, Staining, Immunohistochemistry, Over Expression

    Characterization of an EMT-like phenotype in NICD/p53 −/− primary tumours. ( a ) Staining of the mesenchymal marker N-cadherin and the EMT transcription factors, SNAIL, SLUG and TWIST in NICD/p53 −/− primary tumours. Left panel: N-cadherin is overexpressed in tumour cells invading the desmoplastic area compared with those from the tumour bulk. Scale bars, 40 μm. Middle and right panels: a nuclear overexpression of EMT transcription factors is observed in cancer cells from the desmoplastic area compared with differentiated tubular structures. Scale bars, 100 μm. ( b , c ) Expression of the EMT-inducer ZEB1 in primary tumours of NICD/p53 −/− . ( b ) Left panel: triple immunofluorescence on NICD/p53 −/− primary tumours using 4,6-diamidino-2-phenylindole (DAPI; grey) and GFP staining (green), ECAD (blue) and ZEB1 (red). Scale bars, 40 μm. Right panel: magnification of different tumour cell features coming from the same slide showing that cells undergoing EMT-like processes express the EMT-inducer ZEB1. *Zeb1 negative epithelial tumour cells; ** and *** Zeb1 and GFP-positive, ECAD-negative cells, independently of their morphology. Scale bars, 4 μm. The white arrow labels individual ZEB1-negative cells with a non-epithelial origin. ( c ) The left bar graph shows the frequency of vimentin, SMA or Zeb1-positive cells among GFP-positive cells. Cells were separated in two groups presenting either an epithelial phenotype described by ECAD-positive cells (E+) or cytokeratin-positive cells (CK+), or an EMT-like phenotype characterized by ECAD negative (E−) or CK negative (CK−). The associated tables of contingency ( Supplementary Table 4 ) depict a strong negative correlation (***) between ZEB1 and ECAD expressions ( P

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: Characterization of an EMT-like phenotype in NICD/p53 −/− primary tumours. ( a ) Staining of the mesenchymal marker N-cadherin and the EMT transcription factors, SNAIL, SLUG and TWIST in NICD/p53 −/− primary tumours. Left panel: N-cadherin is overexpressed in tumour cells invading the desmoplastic area compared with those from the tumour bulk. Scale bars, 40 μm. Middle and right panels: a nuclear overexpression of EMT transcription factors is observed in cancer cells from the desmoplastic area compared with differentiated tubular structures. Scale bars, 100 μm. ( b , c ) Expression of the EMT-inducer ZEB1 in primary tumours of NICD/p53 −/− . ( b ) Left panel: triple immunofluorescence on NICD/p53 −/− primary tumours using 4,6-diamidino-2-phenylindole (DAPI; grey) and GFP staining (green), ECAD (blue) and ZEB1 (red). Scale bars, 40 μm. Right panel: magnification of different tumour cell features coming from the same slide showing that cells undergoing EMT-like processes express the EMT-inducer ZEB1. *Zeb1 negative epithelial tumour cells; ** and *** Zeb1 and GFP-positive, ECAD-negative cells, independently of their morphology. Scale bars, 4 μm. The white arrow labels individual ZEB1-negative cells with a non-epithelial origin. ( c ) The left bar graph shows the frequency of vimentin, SMA or Zeb1-positive cells among GFP-positive cells. Cells were separated in two groups presenting either an epithelial phenotype described by ECAD-positive cells (E+) or cytokeratin-positive cells (CK+), or an EMT-like phenotype characterized by ECAD negative (E−) or CK negative (CK−). The associated tables of contingency ( Supplementary Table 4 ) depict a strong negative correlation (***) between ZEB1 and ECAD expressions ( P

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Staining, Marker, Over Expression, Expressing, Immunofluorescence

    An EMT-like phenotype is induced in NICD/p53 −/− primary tumours. ( a , e , i ) Triple immunofluorescence of primary tumours using 4′,6-diamidino-2-phenylindole (grey) and GFP staining (green), an epithelial marker (blue) and a mesenchymal marker (red). Scale bars, 40 μm. b – d , f – h , j – l represent magnifications of the boxes lettered in a , e , i , respectively. ( b , f , j ) Clusters of epithelial cells express GFP and epithelial markers (ECAD, Pan cytokeratin and P120) but do not express mesenchymal markers (vimentin and α-SMA). ( c , d , g , h , k , l ) Single GFP-positive cells, with an epithelial origin, lose epithelial markers and acquire mesenchymal ones irrespective of whether they have epithelial ( c , g , k ) or fibroblast ( d , h , l ) nuclear morphology. Scale bars, 4 μm.

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: An EMT-like phenotype is induced in NICD/p53 −/− primary tumours. ( a , e , i ) Triple immunofluorescence of primary tumours using 4′,6-diamidino-2-phenylindole (grey) and GFP staining (green), an epithelial marker (blue) and a mesenchymal marker (red). Scale bars, 40 μm. b – d , f – h , j – l represent magnifications of the boxes lettered in a , e , i , respectively. ( b , f , j ) Clusters of epithelial cells express GFP and epithelial markers (ECAD, Pan cytokeratin and P120) but do not express mesenchymal markers (vimentin and α-SMA). ( c , d , g , h , k , l ) Single GFP-positive cells, with an epithelial origin, lose epithelial markers and acquire mesenchymal ones irrespective of whether they have epithelial ( c , g , k ) or fibroblast ( d , h , l ) nuclear morphology. Scale bars, 4 μm.

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Immunofluorescence, Staining, Marker

    EMT-like cells are not proliferative. ( a ) Immunohistochemical staining of P21 in NICD/p53 −/− primary tumours, showing nuclear P21 expression in cancer cells at the tumour front. This feature is emphasized in cancer cells invading the desmoplastic area. Scale bars, 100 μm. ( b ) Triple immunofluorescence on NICD/p53 −/− primary tumours using 4′,6-diamidino-2-phenylindole (grey), GFP staining (green), ECAD (blue) and phospho-H3 (red). Scale bars, 40 μm. ( c – e ) Magnification of different tumour cell features coming from the same slide showing some epithelial cells in clusters (c) or fibroblasts (e) in division whether EMT-like cells are quiescent (d). Scale bars, 4 μm. ( f ) Proportion of pH3-positive cells among GFP-positive cells. Among GFP-positive cells, 12% of cells with an epithelial morphology as defined by ECAD positivity (E+; n =585) are proliferative, while only 2% of ECAD-negative cells (E−; n =274) express pH3 (full contingency table indicates dependency χ 2 test, P

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: EMT-like cells are not proliferative. ( a ) Immunohistochemical staining of P21 in NICD/p53 −/− primary tumours, showing nuclear P21 expression in cancer cells at the tumour front. This feature is emphasized in cancer cells invading the desmoplastic area. Scale bars, 100 μm. ( b ) Triple immunofluorescence on NICD/p53 −/− primary tumours using 4′,6-diamidino-2-phenylindole (grey), GFP staining (green), ECAD (blue) and phospho-H3 (red). Scale bars, 40 μm. ( c – e ) Magnification of different tumour cell features coming from the same slide showing some epithelial cells in clusters (c) or fibroblasts (e) in division whether EMT-like cells are quiescent (d). Scale bars, 4 μm. ( f ) Proportion of pH3-positive cells among GFP-positive cells. Among GFP-positive cells, 12% of cells with an epithelial morphology as defined by ECAD positivity (E+; n =585) are proliferative, while only 2% of ECAD-negative cells (E−; n =274) express pH3 (full contingency table indicates dependency χ 2 test, P

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Immunohistochemistry, Staining, Expressing, Immunofluorescence

    A hypothesis on Notch–p53 and Wnt interplay derived from signalling network analysis. Green and grey arrows indicate an activating effect, red arrows depict an inhibiting effect and dotted line indicates an indirect effect. ( a ) Schematic view of three signalling pathways (Wnt, Notch and p53) and the phenotypes of apoptosis, proliferation and EMT induction, showing crosstalk between each other. ( b ) Schematic view at a molecular level showing the involvement of Notch and Wnt pathways, p53 family and microRNAs in activating EMT inducers. The p53 family members (p53, p63 and p73) can be activated by DNA damage and can induce transcription of miRNAs (for example, miR34, miR200 and miR203). These miRNAs target mRNAs coding for β -catenin (Wnt pathway), Notch and the EMT inducers ( Snail, Twist, Slug, Zeb1 and Zeb2 ). Those EMT inducers activate the Wnt pathway that, in turn, activates the Notch pathway, resulting in the activation of Notch (NICD). NICD activates the gene expression of EMT inducers, but can also inhibit transcription of p63 and p73, but not p53. Various signals from tumour microenvironment as extracellular matrix (ECM) components, hormones, growth factors, inflammatory factors and so on (collectively denoted as ‘Tumour microenvironment’ node) can sensitize the activation of EMT programme only in those cells that are in contact with microenvironmental signals.

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: A hypothesis on Notch–p53 and Wnt interplay derived from signalling network analysis. Green and grey arrows indicate an activating effect, red arrows depict an inhibiting effect and dotted line indicates an indirect effect. ( a ) Schematic view of three signalling pathways (Wnt, Notch and p53) and the phenotypes of apoptosis, proliferation and EMT induction, showing crosstalk between each other. ( b ) Schematic view at a molecular level showing the involvement of Notch and Wnt pathways, p53 family and microRNAs in activating EMT inducers. The p53 family members (p53, p63 and p73) can be activated by DNA damage and can induce transcription of miRNAs (for example, miR34, miR200 and miR203). These miRNAs target mRNAs coding for β -catenin (Wnt pathway), Notch and the EMT inducers ( Snail, Twist, Slug, Zeb1 and Zeb2 ). Those EMT inducers activate the Wnt pathway that, in turn, activates the Notch pathway, resulting in the activation of Notch (NICD). NICD activates the gene expression of EMT inducers, but can also inhibit transcription of p63 and p73, but not p53. Various signals from tumour microenvironment as extracellular matrix (ECM) components, hormones, growth factors, inflammatory factors and so on (collectively denoted as ‘Tumour microenvironment’ node) can sensitize the activation of EMT programme only in those cells that are in contact with microenvironmental signals.

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Derivative Assay, Activation Assay, Expressing

    EMT signalling activation in human samples with CRC. ( a – f ) Double immunofluorescence on human samples with CRC using ZEB1 (red) and ECAD (green). ( a ) CRC primary tumour and ( c , e ) liver metastasis, showing ZEB1-positive/ECAD-negative cells invading the desmoplastic area in primary tumour and in liver metastasis. Numbers on a , c , e indicate different cell types observed on the slide as follows: 1 for colorectal primary (in a ) or metastatic cells (in c ), 2 for stroma cells and 3 for hepatocytes. Scale bars, 20 μm. Magnifications from the boxes b,d,f are also represented in b , d , f single 4′,6-diamidino-2-phenylindole, ECAD and ZEB1 staining. ZEB1-positive cells (labelled with an arrowhead) in the liver metastasis are either observed next to colorectal tumour cells ( d ) or surrounded by hepatic tissue ( f ). Scale bars, 4 μm. ( g ) The activity scores computed for the Notch, p53 and Wnt pathways in human colon cancer samples from Tumour Cancer Genome Atlas data set. The data points represent primary tumour samples grouped as non-metastatic (blue) and metastatic (red) according to the observation of distant metastases. The bottom and top of the box correspond to the first and third quartiles of the activity score values, and the band inside the box represents the median. P values are calculated using the two-sample t -test between the two groups.

    Journal: Nature Communications

    Article Title: Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

    doi: 10.1038/ncomms6005

    Figure Lengend Snippet: EMT signalling activation in human samples with CRC. ( a – f ) Double immunofluorescence on human samples with CRC using ZEB1 (red) and ECAD (green). ( a ) CRC primary tumour and ( c , e ) liver metastasis, showing ZEB1-positive/ECAD-negative cells invading the desmoplastic area in primary tumour and in liver metastasis. Numbers on a , c , e indicate different cell types observed on the slide as follows: 1 for colorectal primary (in a ) or metastatic cells (in c ), 2 for stroma cells and 3 for hepatocytes. Scale bars, 20 μm. Magnifications from the boxes b,d,f are also represented in b , d , f single 4′,6-diamidino-2-phenylindole, ECAD and ZEB1 staining. ZEB1-positive cells (labelled with an arrowhead) in the liver metastasis are either observed next to colorectal tumour cells ( d ) or surrounded by hepatic tissue ( f ). Scale bars, 4 μm. ( g ) The activity scores computed for the Notch, p53 and Wnt pathways in human colon cancer samples from Tumour Cancer Genome Atlas data set. The data points represent primary tumour samples grouped as non-metastatic (blue) and metastatic (red) according to the observation of distant metastases. The bottom and top of the box correspond to the first and third quartiles of the activity score values, and the band inside the box represents the median. P values are calculated using the two-sample t -test between the two groups.

    Article Snippet: Antibody references and working dilution were used as follow: ECAD (BD-610182, 1/400), vimentin (CS-5741, 1/300), GFP (ab 13970, 1/300), Pan cytokeratin (Dako ZO622, 1/200), α-SMA (Sigma A2547, 1/600), SLUG (CS-9585, 1/100), SNAIL (ab 85931, 1/100), TWIST (sc-15393, 1/100), ZEB1 (Euromedex 00419, 1/200), β-catenin (BD-610154, 1/50), FASCIN1 (Dako M3567, 1/50), P21 (ab 2961, 1/150), phospho-H3 (Millipore 06570, 1/1000), NICD (sc-6014, 1/100) and HES1 (Clinisciences D134-3, 1/50), P120 (BD-610133, 1/100), P53 (Dako DO-7, 1/200) and N-cadherin (Zymed 3B9, 1/100).

    Techniques: Activation Assay, Immunofluorescence, Staining, Activity Assay

    6-Sho enhances TRAIL-mediated p53 expression. Huh7 cells were treated with 6-sho (5, 10 and 20 µ M) for 18 h. (A) p53 levels assessed by western blot analysis using β-actin as control. (B) Cells stained with p53 antibody (red) and DAPI nuclear stain (blue) evaluated using fluorescence microscopy (magnification, ×400). Western blot analysis of p53 levels in Huh7 cells were pretreated with (C) CQ (10 mM) for 1 h or (D) NAC (10 mM) for 1 h followed by CQ (10 mM) for 1 h prior to exposure to 6-sho (20 µ M) for 18 h and TRAIL (200 ng/ml) for 2 h. 6-Sho, 6-shogaol; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; CQ, chloroquine; p53, tumor-suppressor protein 53; NAC, N -acetyl-L-cysteine.

    Journal: International Journal of Molecular Medicine

    Article Title: Attenuation of autophagy flux by 6-shogaol sensitizes human liver cancer cells to TRAIL-induced apoptosis via p53 and ROS

    doi: 10.3892/ijmm.2018.3994

    Figure Lengend Snippet: 6-Sho enhances TRAIL-mediated p53 expression. Huh7 cells were treated with 6-sho (5, 10 and 20 µ M) for 18 h. (A) p53 levels assessed by western blot analysis using β-actin as control. (B) Cells stained with p53 antibody (red) and DAPI nuclear stain (blue) evaluated using fluorescence microscopy (magnification, ×400). Western blot analysis of p53 levels in Huh7 cells were pretreated with (C) CQ (10 mM) for 1 h or (D) NAC (10 mM) for 1 h followed by CQ (10 mM) for 1 h prior to exposure to 6-sho (20 µ M) for 18 h and TRAIL (200 ng/ml) for 2 h. 6-Sho, 6-shogaol; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; CQ, chloroquine; p53, tumor-suppressor protein 53; NAC, N -acetyl-L-cysteine.

    Article Snippet: Cells were then incubated for 60 min at room temperature with blocking solution (5% FBS in Tris-buffered saline) followed by overnight incubation at 4°C with anti-p62 (1:250; cat. no. PA5-20839; Invitrogen; Thermo Fisher Scientific, Inc.) and anti-p53 (1:250; cat. no. 9286; Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies.

    Techniques: Expressing, Western Blot, Staining, Fluorescence, Microscopy

    Effects of TUN treatment on glioma cell apoptosis. Cells were treated with TUN (2 mg/ml) or vehicle control. (A and B) Apoptosis rate following treatments for (A) BV-2 (A) and (B) BC3H1 cells. (C and D) Western blotting results for expression of cleaved PARP and caspase-9 in (C) BV-2 and (D) BC3H1 (D) cells. (E and F) Western blotting results for expression of Bcl-2 and P53 in (E) BV-2 and (F) BC3H1 cells. **P

    Journal: Oncology Letters

    Article Title: Tunicamycin inhibits progression of glioma cells through downregulation of the MEG-3-regulated wnt/β-catenin signaling pathway

    doi: 10.3892/ol.2018.8416

    Figure Lengend Snippet: Effects of TUN treatment on glioma cell apoptosis. Cells were treated with TUN (2 mg/ml) or vehicle control. (A and B) Apoptosis rate following treatments for (A) BV-2 (A) and (B) BC3H1 cells. (C and D) Western blotting results for expression of cleaved PARP and caspase-9 in (C) BV-2 and (D) BC3H1 (D) cells. (E and F) Western blotting results for expression of Bcl-2 and P53 in (E) BV-2 and (F) BC3H1 cells. **P

    Article Snippet: The purpose protein expression levels were incubated with rabbit anti-mouse primary antibodies: Cyclin D1 (1:500 dilution; cat no. ab18), Cyclin D2 (CDK2; 1:500 dilution; cat no. ab32147), Fibronectin (1:500 dilution; cat no. ab2413), E-cadherin (1:500; ab11512), PRAP1 (1:500; ab52100), Caspase-9 (1:500 dilution; cat no. ab52298), Bcl-2 (1:500 dilution; cat no. ab196495), P53 (1:500 dilution; cat no. ab1431), Wnt (1:500 dilution; cat no. ab15251), β-catenin (1:500 dilution; cat no. ab32572), β-actin (1:2,000 dilution, cat no. ab5694; All antibodies were purchased from Abcam, Cambridge, UK) and then incubated with goat anti-rabbit horseradish peroxidase-labeled immunoglobulin G (1:2,000 dilution, cat no. ab6789, Abcam) for 1 h at 37°C.

    Techniques: Western Blot, Expressing

    Apigenin administration inhibits 22Rv1 tumor growth in athymic nude mice via modulation of p53 signaling pathways. Approximately 1 million cells were injected into both flanks of each mouse to initiate ectopic prostate tumor growth, and apigenin was provided to the animals 2 weeks before cell inoculation. Mice were fed ad libitum with Teklad 8760 autoclaved high-protein diet. Apigenin was provided with 0.5% methyl cellulose and 0.025% Tween 20 as vehicle to these animals perorally on a daily basis. Group I, control, received 0.2 ml vehicle only, Group II received 20 μg apigenin per mouse in 0.2 ml vehicle, and Group III received 50 μg apigenin per mouse in 0.2 ml vehicle daily for 8 weeks. Once the tumor xenografts started growing, their sizes were measured twice weekly in two dimensions throughout the study. (A) Tumor volume (mm 3 ) in control and treated groups. (B) Wet weight of tumors is represented as the mean of 8–10 tumors from each group. (C) Apoptosis as demonstrated by ELISA for 22Rv1 tumors after apigenin intake at the indicated doses. Values are means±SE, n =6–8, repeated twice with similar results. (D) Immunoblots for p53, Ser15-p53, cytochrome c, Bax, Bcl-2, and caspase-3 in tumor lysates after apigenin intake at the indicated doses. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. Significantly different from control: ** p

    Journal: Free radical biology & medicine

    Article Title: Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

    doi: 10.1016/j.freeradbiomed.2008.02.007

    Figure Lengend Snippet: Apigenin administration inhibits 22Rv1 tumor growth in athymic nude mice via modulation of p53 signaling pathways. Approximately 1 million cells were injected into both flanks of each mouse to initiate ectopic prostate tumor growth, and apigenin was provided to the animals 2 weeks before cell inoculation. Mice were fed ad libitum with Teklad 8760 autoclaved high-protein diet. Apigenin was provided with 0.5% methyl cellulose and 0.025% Tween 20 as vehicle to these animals perorally on a daily basis. Group I, control, received 0.2 ml vehicle only, Group II received 20 μg apigenin per mouse in 0.2 ml vehicle, and Group III received 50 μg apigenin per mouse in 0.2 ml vehicle daily for 8 weeks. Once the tumor xenografts started growing, their sizes were measured twice weekly in two dimensions throughout the study. (A) Tumor volume (mm 3 ) in control and treated groups. (B) Wet weight of tumors is represented as the mean of 8–10 tumors from each group. (C) Apoptosis as demonstrated by ELISA for 22Rv1 tumors after apigenin intake at the indicated doses. Values are means±SE, n =6–8, repeated twice with similar results. (D) Immunoblots for p53, Ser15-p53, cytochrome c, Bax, Bcl-2, and caspase-3 in tumor lysates after apigenin intake at the indicated doses. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. Significantly different from control: ** p

    Article Snippet: Antibodies recognizing full-length poly(ADP)ribose polymerase and its cleaved product, p53 (No. 9282); phospho-p53, Ser6 (No. 9285); phospho-p53, Ser9 (No. 9288); phospho-p53, Ser15 (No. 9284); phospho-p53, Ser20 (No. 9287); phospho-p53, Ser37 (No. 9289); phospho-p53, Ser46 (No. 2521); phospho-p53, Ser392 (No. 9281); p14ARF (No. 2407); caspase-3 (No. 9662); caspase-8, 1C12 (No. 9746); and Bcl-XL (No. 2762) were purchased from Cell Signaling Technology (Fremont, CA, USA).

    Techniques: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Western Blot

    Apigenin-induced ROS generation and apoptosis are inhibited by antioxidants and p53 inhibition in 22Rv1 cells. (A) Percentage of cells with high DCF fluorescence in cell culture after 3 h treatment with the indicated concentrations of apigenin, 5 mM NAC, or 25 μM pifithrin-α and their combination. Representative data, means±SE, n =3, from a single experiment, which was repeated twice with similar results. Significantly different from control: ** p

    Journal: Free radical biology & medicine

    Article Title: Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

    doi: 10.1016/j.freeradbiomed.2008.02.007

    Figure Lengend Snippet: Apigenin-induced ROS generation and apoptosis are inhibited by antioxidants and p53 inhibition in 22Rv1 cells. (A) Percentage of cells with high DCF fluorescence in cell culture after 3 h treatment with the indicated concentrations of apigenin, 5 mM NAC, or 25 μM pifithrin-α and their combination. Representative data, means±SE, n =3, from a single experiment, which was repeated twice with similar results. Significantly different from control: ** p

    Article Snippet: Antibodies recognizing full-length poly(ADP)ribose polymerase and its cleaved product, p53 (No. 9282); phospho-p53, Ser6 (No. 9285); phospho-p53, Ser9 (No. 9288); phospho-p53, Ser15 (No. 9284); phospho-p53, Ser20 (No. 9287); phospho-p53, Ser37 (No. 9289); phospho-p53, Ser46 (No. 2521); phospho-p53, Ser392 (No. 9281); p14ARF (No. 2407); caspase-3 (No. 9662); caspase-8, 1C12 (No. 9746); and Bcl-XL (No. 2762) were purchased from Cell Signaling Technology (Fremont, CA, USA).

    Techniques: Inhibition, Fluorescence, Cell Culture

    Effects of apigenin treatment on p53 expression and transcriptional activity in 22Rv1 cells. (A) Immunoblots for p53 and p21/WAF-1 using lysates from 22Rv1 cells treated with various concentrations of apigenin for the indicated time periods. The blot was stripped and reprobed with antiactin antibody to ensure equal protein loading. (B) Densitometric analysis for p53 and p21/WAF-1. (C) Transcriptional activation of p53 using p53-dependent PG-13 promoter. At 24 h posttransfection with the PG-13 luciferase plasmid, 22Rv1 cells were treated with the indicated concentrations of apigenin. Data are means±SE, n =3, repeated twice with similar results. Significantly different from control: ** p

    Journal: Free radical biology & medicine

    Article Title: Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

    doi: 10.1016/j.freeradbiomed.2008.02.007

    Figure Lengend Snippet: Effects of apigenin treatment on p53 expression and transcriptional activity in 22Rv1 cells. (A) Immunoblots for p53 and p21/WAF-1 using lysates from 22Rv1 cells treated with various concentrations of apigenin for the indicated time periods. The blot was stripped and reprobed with antiactin antibody to ensure equal protein loading. (B) Densitometric analysis for p53 and p21/WAF-1. (C) Transcriptional activation of p53 using p53-dependent PG-13 promoter. At 24 h posttransfection with the PG-13 luciferase plasmid, 22Rv1 cells were treated with the indicated concentrations of apigenin. Data are means±SE, n =3, repeated twice with similar results. Significantly different from control: ** p

    Article Snippet: Antibodies recognizing full-length poly(ADP)ribose polymerase and its cleaved product, p53 (No. 9282); phospho-p53, Ser6 (No. 9285); phospho-p53, Ser9 (No. 9288); phospho-p53, Ser15 (No. 9284); phospho-p53, Ser20 (No. 9287); phospho-p53, Ser37 (No. 9289); phospho-p53, Ser46 (No. 2521); phospho-p53, Ser392 (No. 9281); p14ARF (No. 2407); caspase-3 (No. 9662); caspase-8, 1C12 (No. 9746); and Bcl-XL (No. 2762) were purchased from Cell Signaling Technology (Fremont, CA, USA).

    Techniques: Expressing, Activity Assay, Western Blot, Activation Assay, Luciferase, Plasmid Preparation

    Apigenin treatment causes stabilization of p53 in 22Rv1 cells. (A) Immunoblots for p53 at various phosphorylation sites using lysates from 22Rv1 cells treated with various doses of apigenin for 24 h. (B) Immunoblots for p14 ARF and MDM2 using lysates from 22Rv1 cells treated with various doses of apigenin for the indicated time periods. The blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading.

    Journal: Free radical biology & medicine

    Article Title: Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

    doi: 10.1016/j.freeradbiomed.2008.02.007

    Figure Lengend Snippet: Apigenin treatment causes stabilization of p53 in 22Rv1 cells. (A) Immunoblots for p53 at various phosphorylation sites using lysates from 22Rv1 cells treated with various doses of apigenin for 24 h. (B) Immunoblots for p14 ARF and MDM2 using lysates from 22Rv1 cells treated with various doses of apigenin for the indicated time periods. The blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading.

    Article Snippet: Antibodies recognizing full-length poly(ADP)ribose polymerase and its cleaved product, p53 (No. 9282); phospho-p53, Ser6 (No. 9285); phospho-p53, Ser9 (No. 9288); phospho-p53, Ser15 (No. 9284); phospho-p53, Ser20 (No. 9287); phospho-p53, Ser37 (No. 9289); phospho-p53, Ser46 (No. 2521); phospho-p53, Ser392 (No. 9281); p14ARF (No. 2407); caspase-3 (No. 9662); caspase-8, 1C12 (No. 9746); and Bcl-XL (No. 2762) were purchased from Cell Signaling Technology (Fremont, CA, USA).

    Techniques: Western Blot

    Inhibition of p53 causes a decrease in apigenin-mediated apoptosis. (A) Immunoblots for p53 and p21/WAF-1 using lysates from 22Rv1 cells pretreated with 10 μM p53-specific antisense and mismatch oligonucleotides for 8 h, followed by 20 μM apigenin treatment for 16 h and combination of p53-specific antisense oligonucleotide and apigenin for 16 h. (B) Immunoblots for p53 and p21/WAF-1 using lysates from PC-3 (p53 −/− ) and PC-3 (p53 +/+ ) cells treated with 10 μM p53-specific antisense and mismatch oligonucleotides for 8 h, followed by 20 μM apigenin treatment for 16 h and combination of p53-specific antisense oligonucleotide and apigenin for 16 h. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. (C) Apoptosis in the lysates from 22Rv1, PC-3 (p53 −/− ), and PC-3 (p53 +/+ ) cells with the indicated treatments. Values are represented as enrichment factor described under Materials and methods. Data are means±SE, n =3. Significantly different from control: ** p

    Journal: Free radical biology & medicine

    Article Title: Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

    doi: 10.1016/j.freeradbiomed.2008.02.007

    Figure Lengend Snippet: Inhibition of p53 causes a decrease in apigenin-mediated apoptosis. (A) Immunoblots for p53 and p21/WAF-1 using lysates from 22Rv1 cells pretreated with 10 μM p53-specific antisense and mismatch oligonucleotides for 8 h, followed by 20 μM apigenin treatment for 16 h and combination of p53-specific antisense oligonucleotide and apigenin for 16 h. (B) Immunoblots for p53 and p21/WAF-1 using lysates from PC-3 (p53 −/− ) and PC-3 (p53 +/+ ) cells treated with 10 μM p53-specific antisense and mismatch oligonucleotides for 8 h, followed by 20 μM apigenin treatment for 16 h and combination of p53-specific antisense oligonucleotide and apigenin for 16 h. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. (C) Apoptosis in the lysates from 22Rv1, PC-3 (p53 −/− ), and PC-3 (p53 +/+ ) cells with the indicated treatments. Values are represented as enrichment factor described under Materials and methods. Data are means±SE, n =3. Significantly different from control: ** p

    Article Snippet: Antibodies recognizing full-length poly(ADP)ribose polymerase and its cleaved product, p53 (No. 9282); phospho-p53, Ser6 (No. 9285); phospho-p53, Ser9 (No. 9288); phospho-p53, Ser15 (No. 9284); phospho-p53, Ser20 (No. 9287); phospho-p53, Ser37 (No. 9289); phospho-p53, Ser46 (No. 2521); phospho-p53, Ser392 (No. 9281); p14ARF (No. 2407); caspase-3 (No. 9662); caspase-8, 1C12 (No. 9746); and Bcl-XL (No. 2762) were purchased from Cell Signaling Technology (Fremont, CA, USA).

    Techniques: Inhibition, Western Blot

    Apigenin-induced ROS generation, p53 translocation, and cytochrome c release into the cytosol are reversed by treatment with catalase in 22Rv1 cells. (A) Fold change in ROS levels and enrichment factor after 3 h treatment of cells with 1 mM H 2 O 2 or 40 μM apigenin and pretreatment with 400 units/ml recombinant catalase followed by apigenin. Values are represented as means±SE, n =3, from a single experiment, which was repeated twice with similar results. (B) Immunoblots for p53 and p21/WAF-1 after treatment with H 2 O 2 and its combination with catalase. (C) Immunoblots for p53 and p21/WAF-1 after treatment with H 2 O 2 , apigenin, and apigenin combined with catalase. The blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading. (D) Immunoblots for p53 and cytochrome c in the cytosolic and mitochondrial fractions from 22Rv1 cells treated with H 2 O 2 , apigenin, and apigenin combined with catalase. The blot was stripped and reprobed with anti-COX-IV antibody to ensure equal mitochondrial protein loading.

    Journal: Free radical biology & medicine

    Article Title: Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

    doi: 10.1016/j.freeradbiomed.2008.02.007

    Figure Lengend Snippet: Apigenin-induced ROS generation, p53 translocation, and cytochrome c release into the cytosol are reversed by treatment with catalase in 22Rv1 cells. (A) Fold change in ROS levels and enrichment factor after 3 h treatment of cells with 1 mM H 2 O 2 or 40 μM apigenin and pretreatment with 400 units/ml recombinant catalase followed by apigenin. Values are represented as means±SE, n =3, from a single experiment, which was repeated twice with similar results. (B) Immunoblots for p53 and p21/WAF-1 after treatment with H 2 O 2 and its combination with catalase. (C) Immunoblots for p53 and p21/WAF-1 after treatment with H 2 O 2 , apigenin, and apigenin combined with catalase. The blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading. (D) Immunoblots for p53 and cytochrome c in the cytosolic and mitochondrial fractions from 22Rv1 cells treated with H 2 O 2 , apigenin, and apigenin combined with catalase. The blot was stripped and reprobed with anti-COX-IV antibody to ensure equal mitochondrial protein loading.

    Article Snippet: Antibodies recognizing full-length poly(ADP)ribose polymerase and its cleaved product, p53 (No. 9282); phospho-p53, Ser6 (No. 9285); phospho-p53, Ser9 (No. 9288); phospho-p53, Ser15 (No. 9284); phospho-p53, Ser20 (No. 9287); phospho-p53, Ser37 (No. 9289); phospho-p53, Ser46 (No. 2521); phospho-p53, Ser392 (No. 9281); p14ARF (No. 2407); caspase-3 (No. 9662); caspase-8, 1C12 (No. 9746); and Bcl-XL (No. 2762) were purchased from Cell Signaling Technology (Fremont, CA, USA).

    Techniques: Translocation Assay, Recombinant, Western Blot

    Apigenin treatment causes ROS generation, disruption of mitochondrial membrane potential, translocation of p53 to the mitochondria, and cytochrome c release into the cytosol in 22Rv1 cells. (A) Percentage of cells with high DCF fluorescence in cell culture after 3 h treatment with the indicated concentrations of apigenin. Representative data, means±SE, n =3, from a single experiment, which was repeated twice with similar results. Significantly different from control: ** p

    Journal: Free radical biology & medicine

    Article Title: Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation

    doi: 10.1016/j.freeradbiomed.2008.02.007

    Figure Lengend Snippet: Apigenin treatment causes ROS generation, disruption of mitochondrial membrane potential, translocation of p53 to the mitochondria, and cytochrome c release into the cytosol in 22Rv1 cells. (A) Percentage of cells with high DCF fluorescence in cell culture after 3 h treatment with the indicated concentrations of apigenin. Representative data, means±SE, n =3, from a single experiment, which was repeated twice with similar results. Significantly different from control: ** p

    Article Snippet: Antibodies recognizing full-length poly(ADP)ribose polymerase and its cleaved product, p53 (No. 9282); phospho-p53, Ser6 (No. 9285); phospho-p53, Ser9 (No. 9288); phospho-p53, Ser15 (No. 9284); phospho-p53, Ser20 (No. 9287); phospho-p53, Ser37 (No. 9289); phospho-p53, Ser46 (No. 2521); phospho-p53, Ser392 (No. 9281); p14ARF (No. 2407); caspase-3 (No. 9662); caspase-8, 1C12 (No. 9746); and Bcl-XL (No. 2762) were purchased from Cell Signaling Technology (Fremont, CA, USA).

    Techniques: Translocation Assay, Fluorescence, Cell Culture

    Model for SC-driven epidermal tumour initiation by mutant Lef1. Expression of mutant Lef1 results in defective DNA damage response and impairment of SC-specific surveillance mechanisms, including p53 activation. SCs γH2AX carrying high levels of DNA damage undergo apoptosis, mainly by blocking the normal Bcl-2 response. Bulge SCs (BSCs) with lower degree of damaged DNA escape normal control of SC proliferation to maintain the epidermal tissue. Consequently, these proliferative SCs carrying DNA breaks accumulate further mutations, thereby initiating tumour formation.

    Journal: Nature Communications

    Article Title: Interfering with stem cell-specific gatekeeper functions controls tumour initiation and malignant progression of skin tumours

    doi: 10.1038/ncomms6874

    Figure Lengend Snippet: Model for SC-driven epidermal tumour initiation by mutant Lef1. Expression of mutant Lef1 results in defective DNA damage response and impairment of SC-specific surveillance mechanisms, including p53 activation. SCs γH2AX carrying high levels of DNA damage undergo apoptosis, mainly by blocking the normal Bcl-2 response. Bulge SCs (BSCs) with lower degree of damaged DNA escape normal control of SC proliferation to maintain the epidermal tissue. Consequently, these proliferative SCs carrying DNA breaks accumulate further mutations, thereby initiating tumour formation.

    Article Snippet: Membranes were probed with antibodies against p53 (1:2,000, Cell Signaling), p-p53 (1:2,000, Cell Signaling) and GapDH (1:2,000, Abcam) followed by incubation with anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (1:5,000, GE Healthcare).

    Techniques: Mutagenesis, Expressing, Activation Assay, Blocking Assay

    Defective p53 response in human sebaceous tumours and SCs of mutant Lef1 mice. ( a – c ) Immunofluorescent staining of p53 (red) and K14 (green) in SCC ( n =2 mice, 3 tumours; a , arrows), sebaceous tumour of K15ΔNLef1 mice ( n =4 mice, 10 tumours) ( b ) and human sebaceous tumour ( c ; n =7). ( d – g ) Immunofluorescent detection of p53 in HF bulge of wild-type ( d , e ) and K15ΔNLef1 mice ( f , g ) without ( d , f ) and following UV irradiation ( e , g ; n =3 mice). Nuclei stained in red (PI). ( h ) Quantification of p53 detection in HF of wild-type and K15ΔNLef1 mice following UV treatments ( n =6 mice). Significance was calculated by Student’s t -test (*** P

    Journal: Nature Communications

    Article Title: Interfering with stem cell-specific gatekeeper functions controls tumour initiation and malignant progression of skin tumours

    doi: 10.1038/ncomms6874

    Figure Lengend Snippet: Defective p53 response in human sebaceous tumours and SCs of mutant Lef1 mice. ( a – c ) Immunofluorescent staining of p53 (red) and K14 (green) in SCC ( n =2 mice, 3 tumours; a , arrows), sebaceous tumour of K15ΔNLef1 mice ( n =4 mice, 10 tumours) ( b ) and human sebaceous tumour ( c ; n =7). ( d – g ) Immunofluorescent detection of p53 in HF bulge of wild-type ( d , e ) and K15ΔNLef1 mice ( f , g ) without ( d , f ) and following UV irradiation ( e , g ; n =3 mice). Nuclei stained in red (PI). ( h ) Quantification of p53 detection in HF of wild-type and K15ΔNLef1 mice following UV treatments ( n =6 mice). Significance was calculated by Student’s t -test (*** P

    Article Snippet: Membranes were probed with antibodies against p53 (1:2,000, Cell Signaling), p-p53 (1:2,000, Cell Signaling) and GapDH (1:2,000, Abcam) followed by incubation with anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (1:5,000, GE Healthcare).

    Techniques: Mutagenesis, Mouse Assay, Staining, Irradiation

    Venn diagram depicting overlap between up-regulated and down-regulated genes when comparing homozygous mutant tp53M214K/M214K and tp53 del/del MPNST to whole adult zebrafish.

    Journal: eLife

    Article Title: tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish

    doi: 10.7554/eLife.37202

    Figure Lengend Snippet: Venn diagram depicting overlap between up-regulated and down-regulated genes when comparing homozygous mutant tp53M214K/M214K and tp53 del/del MPNST to whole adult zebrafish.

    Article Snippet: Western blot was performed using anti-tp53 (ab77813, Abcam) and anti-actin (A2066, Sigma) antibodies.

    Techniques: Mutagenesis

    tp53 del/del tumors efficiently transplant into syngeneic CG1 strain zebrafish. ( A–E ) A primary tp53 del/del MPNSTs that formed in the eye transplanted orthotopically into the periocular space ( A–C ) or into the peritoneum of CG1-strain recipient fish ( D–E ). Intraperitoneal injection (i/p). ( F–I ) tp53 del/del Tg( ubi :GFP)-positive angiosarcoma. Primary tumor-bearing fish ( F–G ) and transplanted animal ( H–I ). ( J–R ) tp53 del/del Tg( ubi :GFP)-positive leukemia. Primary leukemia ( J–K ) and transplanted leukemia shown at 20 days post-transplantation ( L–R ). Whole kidney marrow was isolated from leukemia-engrafted fish and analyzed by FACS ( N–O ). ( N ) Forward and side scatter plot of whole kidney marrow of unlabeled CG1 host animal to assess ubi: GFP-positive tp53 del/del leukemia cells following transplantation. ( O ) Analysis of GFP+ ubi: GFP-positive tp53 del/del leukemia cells following FACS. Purity was ≥90%. ( P–R ) Cytospins and Wright/Giemsa staining of whole kidney marrow cells isolated from wildtype fish ( P ) compared with FACS sorted GFP+ cells from two representative aggressive NK cell-like leukemias, showing large blastic cells with abundant basophilic, vacuolated cytoplasm ( Q–R ). ( S–V ) Embryonal rhabdomyosarcoma arising in tp53 del/del fish micro-injected at the one-cell stage with linearized rag2 :kRAS G12D + rag2 :GFP. Primary ( S ), transplanted (2°) ( T ), and serially transplanted ERMS (3°) ( U,V ). Whole animal bright-field images ( A,D,F,J ) and merged GFP-fluorescence images ( G,H,K,L,S–U ). Hematoxylin and eosin stained sections of engrafted tumors ( B–C,E, I, M,V ). Scale bars are 5 mm in whole animal images and 100 μm for histology images.

    Journal: eLife

    Article Title: tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish

    doi: 10.7554/eLife.37202

    Figure Lengend Snippet: tp53 del/del tumors efficiently transplant into syngeneic CG1 strain zebrafish. ( A–E ) A primary tp53 del/del MPNSTs that formed in the eye transplanted orthotopically into the periocular space ( A–C ) or into the peritoneum of CG1-strain recipient fish ( D–E ). Intraperitoneal injection (i/p). ( F–I ) tp53 del/del Tg( ubi :GFP)-positive angiosarcoma. Primary tumor-bearing fish ( F–G ) and transplanted animal ( H–I ). ( J–R ) tp53 del/del Tg( ubi :GFP)-positive leukemia. Primary leukemia ( J–K ) and transplanted leukemia shown at 20 days post-transplantation ( L–R ). Whole kidney marrow was isolated from leukemia-engrafted fish and analyzed by FACS ( N–O ). ( N ) Forward and side scatter plot of whole kidney marrow of unlabeled CG1 host animal to assess ubi: GFP-positive tp53 del/del leukemia cells following transplantation. ( O ) Analysis of GFP+ ubi: GFP-positive tp53 del/del leukemia cells following FACS. Purity was ≥90%. ( P–R ) Cytospins and Wright/Giemsa staining of whole kidney marrow cells isolated from wildtype fish ( P ) compared with FACS sorted GFP+ cells from two representative aggressive NK cell-like leukemias, showing large blastic cells with abundant basophilic, vacuolated cytoplasm ( Q–R ). ( S–V ) Embryonal rhabdomyosarcoma arising in tp53 del/del fish micro-injected at the one-cell stage with linearized rag2 :kRAS G12D + rag2 :GFP. Primary ( S ), transplanted (2°) ( T ), and serially transplanted ERMS (3°) ( U,V ). Whole animal bright-field images ( A,D,F,J ) and merged GFP-fluorescence images ( G,H,K,L,S–U ). Hematoxylin and eosin stained sections of engrafted tumors ( B–C,E, I, M,V ). Scale bars are 5 mm in whole animal images and 100 μm for histology images.

    Article Snippet: Western blot was performed using anti-tp53 (ab77813, Abcam) and anti-actin (A2066, Sigma) antibodies.

    Techniques: Fluorescence In Situ Hybridization, Injection, Transplantation Assay, Isolation, FACS, Staining, Fluorescence

    tp53 del/del kRAS G12D - induced ERMS have increased invasion and metastasis. ( A–F ) Whole animal fluorescent images of CG1-strain fish engrafted into the dorsolateral musculature with non-disseminated ( A–C ) and disseminated ERMS ( D–F ). Days post transplantation (dpt). White lines demarcate GFP+ tumor area. White arrowheads show site of injection and yellow arrowheads denote metastatic lesions. ( G ) H and E and ( H ) GFP immunohistological staining of fish engrafted with metastatic tp53 del/del kRAS G12D -induced ERMS. ( I ) Quantification of growth confined to site of injection (green bars) and compared with animals that exhibited local invasion or metastatic ERMS following tumor engraftment until fish were moribund. X-axis identifies 5 tp53 wt/wt and 11 tp53 del/del ERMS primary tumors that were transplanted into wild-type CG1 syngeneic host zebrafish. p=0.003, one-sided Fisher’s exact test. Scale bars denote 5 mm.

    Journal: eLife

    Article Title: tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish

    doi: 10.7554/eLife.37202

    Figure Lengend Snippet: tp53 del/del kRAS G12D - induced ERMS have increased invasion and metastasis. ( A–F ) Whole animal fluorescent images of CG1-strain fish engrafted into the dorsolateral musculature with non-disseminated ( A–C ) and disseminated ERMS ( D–F ). Days post transplantation (dpt). White lines demarcate GFP+ tumor area. White arrowheads show site of injection and yellow arrowheads denote metastatic lesions. ( G ) H and E and ( H ) GFP immunohistological staining of fish engrafted with metastatic tp53 del/del kRAS G12D -induced ERMS. ( I ) Quantification of growth confined to site of injection (green bars) and compared with animals that exhibited local invasion or metastatic ERMS following tumor engraftment until fish were moribund. X-axis identifies 5 tp53 wt/wt and 11 tp53 del/del ERMS primary tumors that were transplanted into wild-type CG1 syngeneic host zebrafish. p=0.003, one-sided Fisher’s exact test. Scale bars denote 5 mm.

    Article Snippet: Western blot was performed using anti-tp53 (ab77813, Abcam) and anti-actin (A2066, Sigma) antibodies.

    Techniques: Fluorescence In Situ Hybridization, Transplantation Assay, Injection, Staining

    Homozygous tp53 del/del zebrafish spontaneously develop a wide range of tumor types. ( A ) tp53 genomic locus and CG1 tp53 del/del allele. TALEN arms were designed to target the 5’ and 3’ genomic sequence of tp53 (red). ( B–M ) CG1 tp53 del/del zebrafish develop leukemia ( B–D ), angiosarcoma ( E–G ), MPNSTs ( H–J ), and germ cell tumors ( K–M ). Whole animal images ( B,E,H,K ), hematoxylin/eosin (H and E) stained sections ( C,D,F,G,I,L,M ), and immunohistochemistry for Sox10 ( J ). Blast-like leukemia cells predominate in the kidney marrow and efface the renal tubules (black arrow, ( D ). ( N ) Tumor incidence in CG1 tp53 del/del zebrafish (n = 134). ( O ) Quantitation of tumor types that form in CG1 tp53 del/del mutant zebrafish by 55 weeks of life based on histology review (n = 51). ( P–S ) kRAS G12D -induced embryonal rhabdomyosarcoma (ERMS) generated in CG1 tp53 del/del zebrafish. Whole animal bright field and GFP-epifluorescence overlap images ( P and Q , respectively). H and E stained sections revealed features consistent with human ERMS ( R,S ). Scale bars equal 12.5 mm in whole animal images and 100 μm in histology images.

    Journal: eLife

    Article Title: tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish

    doi: 10.7554/eLife.37202

    Figure Lengend Snippet: Homozygous tp53 del/del zebrafish spontaneously develop a wide range of tumor types. ( A ) tp53 genomic locus and CG1 tp53 del/del allele. TALEN arms were designed to target the 5’ and 3’ genomic sequence of tp53 (red). ( B–M ) CG1 tp53 del/del zebrafish develop leukemia ( B–D ), angiosarcoma ( E–G ), MPNSTs ( H–J ), and germ cell tumors ( K–M ). Whole animal images ( B,E,H,K ), hematoxylin/eosin (H and E) stained sections ( C,D,F,G,I,L,M ), and immunohistochemistry for Sox10 ( J ). Blast-like leukemia cells predominate in the kidney marrow and efface the renal tubules (black arrow, ( D ). ( N ) Tumor incidence in CG1 tp53 del/del zebrafish (n = 134). ( O ) Quantitation of tumor types that form in CG1 tp53 del/del mutant zebrafish by 55 weeks of life based on histology review (n = 51). ( P–S ) kRAS G12D -induced embryonal rhabdomyosarcoma (ERMS) generated in CG1 tp53 del/del zebrafish. Whole animal bright field and GFP-epifluorescence overlap images ( P and Q , respectively). H and E stained sections revealed features consistent with human ERMS ( R,S ). Scale bars equal 12.5 mm in whole animal images and 100 μm in histology images.

    Article Snippet: Western blot was performed using anti-tp53 (ab77813, Abcam) and anti-actin (A2066, Sigma) antibodies.

    Techniques: Sequencing, Staining, Immunohistochemistry, Quantitation Assay, Mutagenesis, Generated

    p53 restoration induces senescence in premalignant cyclin D1-expressing pineal cells. (A and B) Shown are representative staining for BrdU along with the corresponding DAPI nuclear stain (A) and senescence-associated beta-galactosidase (SABG) staining (B) in Irbp-CyclinD1 , p53ERTAM Ki /− pineal cells explanted at P10 and treated for 7 days with either vehicle (Vh) or 4OHT to restore p53, as indicated. The bottom panels show percent BrdU-positive cells (A) and SABG-positive area (measured in pixel density, as explained in Materials and Methods) (B) under each condition, as indicated. Each point represents the means from 3 independent experiments. (C) Representative immunostaining for the indicated proteins in pineal gland sections from Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− mice treated for 10 days (P60 to P70) with Vh or tamoxifen (TAM) and, as controls, Irbp-Cyclin D1 , p53 −/− mice treated with TAM (p53−/− + TAM). (D) Number of Ki67-positive cells per field under each condition shown in panel C as indicated. Each point represents the means using at least 4 pineal glands. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: p53 restoration induces senescence in premalignant cyclin D1-expressing pineal cells. (A and B) Shown are representative staining for BrdU along with the corresponding DAPI nuclear stain (A) and senescence-associated beta-galactosidase (SABG) staining (B) in Irbp-CyclinD1 , p53ERTAM Ki /− pineal cells explanted at P10 and treated for 7 days with either vehicle (Vh) or 4OHT to restore p53, as indicated. The bottom panels show percent BrdU-positive cells (A) and SABG-positive area (measured in pixel density, as explained in Materials and Methods) (B) under each condition, as indicated. Each point represents the means from 3 independent experiments. (C) Representative immunostaining for the indicated proteins in pineal gland sections from Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− mice treated for 10 days (P60 to P70) with Vh or tamoxifen (TAM) and, as controls, Irbp-Cyclin D1 , p53 −/− mice treated with TAM (p53−/− + TAM). (D) Number of Ki67-positive cells per field under each condition shown in panel C as indicated. Each point represents the means using at least 4 pineal glands. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Expressing, Staining, Immunostaining, Mouse Assay

    p53 restoration is not effective in suppressing proliferation of malignant pineal tumor cells unless combined with genotoxic stress. (A) Representative hematoxylin and eosin staining (H E) and Ki67 immunostaining in Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− pineal tumors after 6 days of treatment with tamoxifen (TAM) to restore p53. (B) Representative staining for SABG in explanted Irbp-Cyclin D1 , p53ERTAM Ki /− pineal tumor cells treated for 7 days with vehicle (Vh), 4OHT to restore p53, etoposide to induce genotoxic stress (Etop), or etoposide and 4OHT (Etop + 4OHT), as indicated. (C) Percent SABG-positive area under the conditions described for panel B, where area was measured in pixel density as explained in Materials and Methods. (D) Representative BrdU level and corresponding DAPI nuclear staining under the same conditions as those for panel B. (E) Percent BrdU-positive cells for the conditions depicted in panel D. (F) Representative TUNEL staining to detect apoptosis, under the same conditions as those for panel B, at 48 h after treatment. (G) Percent TUNEL-positive cells under the same conditions as those for panel F. (H, upper) Quantitation of the total number of cells under the conditions described for panel B at 7 days after treatment and normalized to the vehicle-treated control. (Lower) Cresyl violet stain for colony formation assay under the same conditions at 14 days after treatment. Each point in panels C, E, G, and H represents the means from at least 5 fields and is representative of at least 2 independent experiments. Bars represent standard deviations, asterisks denote a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: p53 restoration is not effective in suppressing proliferation of malignant pineal tumor cells unless combined with genotoxic stress. (A) Representative hematoxylin and eosin staining (H E) and Ki67 immunostaining in Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− pineal tumors after 6 days of treatment with tamoxifen (TAM) to restore p53. (B) Representative staining for SABG in explanted Irbp-Cyclin D1 , p53ERTAM Ki /− pineal tumor cells treated for 7 days with vehicle (Vh), 4OHT to restore p53, etoposide to induce genotoxic stress (Etop), or etoposide and 4OHT (Etop + 4OHT), as indicated. (C) Percent SABG-positive area under the conditions described for panel B, where area was measured in pixel density as explained in Materials and Methods. (D) Representative BrdU level and corresponding DAPI nuclear staining under the same conditions as those for panel B. (E) Percent BrdU-positive cells for the conditions depicted in panel D. (F) Representative TUNEL staining to detect apoptosis, under the same conditions as those for panel B, at 48 h after treatment. (G) Percent TUNEL-positive cells under the same conditions as those for panel F. (H, upper) Quantitation of the total number of cells under the conditions described for panel B at 7 days after treatment and normalized to the vehicle-treated control. (Lower) Cresyl violet stain for colony formation assay under the same conditions at 14 days after treatment. Each point in panels C, E, G, and H represents the means from at least 5 fields and is representative of at least 2 independent experiments. Bars represent standard deviations, asterisks denote a statistically significant difference ( P

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Staining, Immunostaining, TUNEL Assay, Quantitation Assay, Colony Assay

    p53 gene and pathway likely are not active in human sPNET. (A) Results of sequencing of p53 exons 4 to 11 in 6 human sPNET samples. (B) FISH staining for p53 gene deletion in tumor 3. The inset shows a positive control. (C) Quantitation of the intensity of expression of p53, p21 CIP1 , p14 ARF , and HDM2, detected by immunohistochemistry, in sPNET samples. (D) Representative images of immunostaining for the indicated proteins in human sPNET samples. The insets represent the respective positive controls. (E) Analysis of p14 Arf promoter methylation status in sPNET samples (T1, T2, T3, and T4), showing unmethylated (U) and methylated (M) sequences. NC, negative control; PC, positive control.

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: p53 gene and pathway likely are not active in human sPNET. (A) Results of sequencing of p53 exons 4 to 11 in 6 human sPNET samples. (B) FISH staining for p53 gene deletion in tumor 3. The inset shows a positive control. (C) Quantitation of the intensity of expression of p53, p21 CIP1 , p14 ARF , and HDM2, detected by immunohistochemistry, in sPNET samples. (D) Representative images of immunostaining for the indicated proteins in human sPNET samples. The insets represent the respective positive controls. (E) Analysis of p14 Arf promoter methylation status in sPNET samples (T1, T2, T3, and T4), showing unmethylated (U) and methylated (M) sequences. NC, negative control; PC, positive control.

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Sequencing, Fluorescence In Situ Hybridization, Staining, Positive Control, Quantitation Assay, Expressing, Immunohistochemistry, Immunostaining, Methylation, Negative Control

    p53 is necessary for maintenance of RasV12-induced senescence in fibroblasts. (A) Representative staining for SABG in p53ERTAM Ki /− MEFs transduced with RasV12 and treated with either vehicle (Vh) or 4-OH tamoxifen (4OHT) to restore p53, as indicated. (B) Percent SABG-positive cells under the conditions represented in panel A. (C) BrdU and corresponding DAPI nuclear stain in p53ERTAM Ki /− MEFs treated as described for panel A. (D) Percent BrdU-positive cells under the conditions represented in panel C. (E) Representative staining for BrdU and corresponding DAPI nuclear stain in p53ERTAM Ki /− MEFs after RasV12 transduction and treatment with 4OHT to restore p53 for 1 week. After this, 4OHT treatment continued (4OHT) or was withdrawn to inactivate p53 (4OHT/OFF) for another week. (F) Percent BrdU-positive cells under the conditions represented in panel E. (G) Western blotting for the indicated proteins in p53ERTAM Ki /− MEFs after RasV12 transduction and treatment with 4OHT to restore p53 for 2 weeks (senescent cells [S]) or treatment with 4OHT for 1 week and then withdrawal to inactivate p53 for another week (S-p53OFF) or in control, RasV12 -transduced, vehicle-treated MEFs as never-senescent controls (NS). (H) Representative staining for SABG in p53ERTAM Ki /− MEFs treated as described for panel E. (I) Percent SABG-positive cells under the conditions represented in panel H. (J and K) Cell density assay by Cresyl violet staining (J) and soft-agar colony formation assay (K) in RasV12 -transduced p53ERTAM Ki /− MEFs after treatment with 4OHT to restore p53 for 1 week. Treatment was continued (4OHT) or was withdrawn to inactivate p53 (4OHT/OFF) for another 2 weeks, as indicated. (L) Mean number of colonies per field under each condition shown in panel K. Each point in panels B, D, F, I, and L represents the means from at least 5 fields and is representative of at least 2 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: p53 is necessary for maintenance of RasV12-induced senescence in fibroblasts. (A) Representative staining for SABG in p53ERTAM Ki /− MEFs transduced with RasV12 and treated with either vehicle (Vh) or 4-OH tamoxifen (4OHT) to restore p53, as indicated. (B) Percent SABG-positive cells under the conditions represented in panel A. (C) BrdU and corresponding DAPI nuclear stain in p53ERTAM Ki /− MEFs treated as described for panel A. (D) Percent BrdU-positive cells under the conditions represented in panel C. (E) Representative staining for BrdU and corresponding DAPI nuclear stain in p53ERTAM Ki /− MEFs after RasV12 transduction and treatment with 4OHT to restore p53 for 1 week. After this, 4OHT treatment continued (4OHT) or was withdrawn to inactivate p53 (4OHT/OFF) for another week. (F) Percent BrdU-positive cells under the conditions represented in panel E. (G) Western blotting for the indicated proteins in p53ERTAM Ki /− MEFs after RasV12 transduction and treatment with 4OHT to restore p53 for 2 weeks (senescent cells [S]) or treatment with 4OHT for 1 week and then withdrawal to inactivate p53 for another week (S-p53OFF) or in control, RasV12 -transduced, vehicle-treated MEFs as never-senescent controls (NS). (H) Representative staining for SABG in p53ERTAM Ki /− MEFs treated as described for panel E. (I) Percent SABG-positive cells under the conditions represented in panel H. (J and K) Cell density assay by Cresyl violet staining (J) and soft-agar colony formation assay (K) in RasV12 -transduced p53ERTAM Ki /− MEFs after treatment with 4OHT to restore p53 for 1 week. Treatment was continued (4OHT) or was withdrawn to inactivate p53 (4OHT/OFF) for another 2 weeks, as indicated. (L) Mean number of colonies per field under each condition shown in panel K. Each point in panels B, D, F, I, and L represents the means from at least 5 fields and is representative of at least 2 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Staining, Transduction, Western Blot, Soft Agar Assay

    4OHT restores p53 activity in p53ER(TAM) cells and has no effect on explanted Irbp-Cyclin D1 , p53 −/− tumor cells. (A) Western blotting for the indicated proteins in wild-type MEFs (WT MEF) as controls and in p53ERTAM pineal tumor cells treated for 48 h with 10 μM etoposide (Etop) or vehicle and with 4-hydroxytamoxifen (4OHT) or vehicle. GAPDH was used as a loading control. (B, D, and F) Representative staining for SABG (B) and BrdU (D, lower), and the corresponding DAPI nuclear stain (D, upper), at 7 days after treatment and TUNEL staining (F, lower) and the corresponding DAPI nuclear stain (upper) at 48 h after treatment in explanted Irbp-Cyclin D1 , p53 −/− pineal tumor cells treated with vehicle (Vh), 4OHT, etoposide, or both (Etop + 4OHT), as indicated. (C, E, and G) Percentages of SABG-positive area (C), BrdU-positive cells (E), and TUNEL-positive cells (G) under the conditions shown in panels B, D, and F, respectively. Each point represents the means from at least 5 fields and is representative of 2 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: 4OHT restores p53 activity in p53ER(TAM) cells and has no effect on explanted Irbp-Cyclin D1 , p53 −/− tumor cells. (A) Western blotting for the indicated proteins in wild-type MEFs (WT MEF) as controls and in p53ERTAM pineal tumor cells treated for 48 h with 10 μM etoposide (Etop) or vehicle and with 4-hydroxytamoxifen (4OHT) or vehicle. GAPDH was used as a loading control. (B, D, and F) Representative staining for SABG (B) and BrdU (D, lower), and the corresponding DAPI nuclear stain (D, upper), at 7 days after treatment and TUNEL staining (F, lower) and the corresponding DAPI nuclear stain (upper) at 48 h after treatment in explanted Irbp-Cyclin D1 , p53 −/− pineal tumor cells treated with vehicle (Vh), 4OHT, etoposide, or both (Etop + 4OHT), as indicated. (C, E, and G) Percentages of SABG-positive area (C), BrdU-positive cells (E), and TUNEL-positive cells (G) under the conditions shown in panels B, D, and F, respectively. Each point represents the means from at least 5 fields and is representative of 2 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Activity Assay, Western Blot, Staining, TUNEL Assay

    Tamoxifen treatment restores p53 activity in pineal glands in vivo . (A) Representative immunostaining for p21 Cip1 in pineal gland sections and spleen of Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− mice that underwent irradiation after treatment for 6 days (P10 to P16) with vehicle (Vh) or tamoxifen (TAM) as indicated. (B) Number of p21-positive cells per field under the conditions shown in panel A. Each point represents the means from 2 independent experiments. Bars represent standard deviations, and an asterisk denotes a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: Tamoxifen treatment restores p53 activity in pineal glands in vivo . (A) Representative immunostaining for p21 Cip1 in pineal gland sections and spleen of Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− mice that underwent irradiation after treatment for 6 days (P10 to P16) with vehicle (Vh) or tamoxifen (TAM) as indicated. (B) Number of p21-positive cells per field under the conditions shown in panel A. Each point represents the means from 2 independent experiments. Bars represent standard deviations, and an asterisk denotes a statistically significant difference ( P

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Activity Assay, In Vivo, Immunostaining, Mouse Assay, Irradiation

    Mdm2 but not p19 Arf expression levels correlate with p53 restoration efficacy in pineal tumors. (A) qRT-PCR analysis of mRNA expression levels of p19Arf relative to those of GAPDH in Irbp-Cyclin D1 , p53 +/+ pineal glands at P10 (proliferating, presenescent) or P49 (senescent) and Irbp-Cyclin D1 , p53 −/− pineal glands at the pretumorigenic (P49) or tumor (Tumor) stage, as indicated. Each point represents the means from 3 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: Mdm2 but not p19 Arf expression levels correlate with p53 restoration efficacy in pineal tumors. (A) qRT-PCR analysis of mRNA expression levels of p19Arf relative to those of GAPDH in Irbp-Cyclin D1 , p53 +/+ pineal glands at P10 (proliferating, presenescent) or P49 (senescent) and Irbp-Cyclin D1 , p53 −/− pineal glands at the pretumorigenic (P49) or tumor (Tumor) stage, as indicated. Each point represents the means from 3 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Expressing, Quantitative RT-PCR

    p53 is needed for the maintenance of the senescence-like response in cyclin D1-expressing pineal cells. (A) Representative immunostaining for Ki67 in pineal gland sections of Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− mice treated for 10 days (P60 to P70) with TAM to restore p53 and then either treated further with tamoxifen (TAM) or withdrawn from treatment (TAM/OFF) to inactivate p53 for another 10 days, as indicated. (B) Mean number of Ki67-positive cells per field under the conditions shown in panel A. Each point represents the means from at least 5 fields and is representative of 2 independent experiments. (C) Representative immunostaining for the indicated markers of senescence under the same conditions as those for panel A. (D) Representative hematoxylin and eosin staining of Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− pineal glands from mice treated for 10 days (P60 to P70) with vehicle (Vh) and from mice that were treated for 10 days with tamoxifen to restore p53 and then either treated further with tamoxifen or that had tamoxifen withdrawn to inactivate p53 (TAM/OFF) for another 10 days, as indicated. (E) Age at clinical tumor formation in a cohort of mice treated with vehicle, tamoxifen for 1 month from P60 to P90 (TAM/OFF), or continuous tamoxifen treatment, as indicated. (F and H) Representative staining for SABG (F) and BrdU (H, left) and corresponding DAPI nuclear stain (H, right) in Irbp-CyclinD1 , p53ERTAM Ki /− pineal cells explanted at P10. The cells were treated for 7 days with 4OHT to restore p53 and then either treated further with 4OHT (4OHT) or withdrawn from tamoxifen treatment to inactivate p53 (4OHT/OFF) for another 7 days as indicated. (G and I) Percentages of SABG-positive area (G) and BrdU-positive cells (I), under the conditions shown in panel F and H, respectively, are shown. Each point represents the means from at least 5 fields and is representative of 2 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Journal: Molecular and Cellular Biology

    Article Title: p53 Restoration in Induction and Maintenance of Senescence: Differential Effects in Premalignant and Malignant Tumor Cells

    doi: 10.1128/MCB.00747-15

    Figure Lengend Snippet: p53 is needed for the maintenance of the senescence-like response in cyclin D1-expressing pineal cells. (A) Representative immunostaining for Ki67 in pineal gland sections of Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− mice treated for 10 days (P60 to P70) with TAM to restore p53 and then either treated further with tamoxifen (TAM) or withdrawn from treatment (TAM/OFF) to inactivate p53 for another 10 days, as indicated. (B) Mean number of Ki67-positive cells per field under the conditions shown in panel A. Each point represents the means from at least 5 fields and is representative of 2 independent experiments. (C) Representative immunostaining for the indicated markers of senescence under the same conditions as those for panel A. (D) Representative hematoxylin and eosin staining of Irbp-Cyclin D1 , p53ER ( TAM ) Ki /− pineal glands from mice treated for 10 days (P60 to P70) with vehicle (Vh) and from mice that were treated for 10 days with tamoxifen to restore p53 and then either treated further with tamoxifen or that had tamoxifen withdrawn to inactivate p53 (TAM/OFF) for another 10 days, as indicated. (E) Age at clinical tumor formation in a cohort of mice treated with vehicle, tamoxifen for 1 month from P60 to P90 (TAM/OFF), or continuous tamoxifen treatment, as indicated. (F and H) Representative staining for SABG (F) and BrdU (H, left) and corresponding DAPI nuclear stain (H, right) in Irbp-CyclinD1 , p53ERTAM Ki /− pineal cells explanted at P10. The cells were treated for 7 days with 4OHT to restore p53 and then either treated further with 4OHT (4OHT) or withdrawn from tamoxifen treatment to inactivate p53 (4OHT/OFF) for another 7 days as indicated. (G and I) Percentages of SABG-positive area (G) and BrdU-positive cells (I), under the conditions shown in panel F and H, respectively, are shown. Each point represents the means from at least 5 fields and is representative of 2 independent experiments. Bars represent standard deviations, and asterisks denote a statistically significant difference ( P

    Article Snippet: Electrophoresis was performed using 12% Tris-chloride gels and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA), blocked with 5% nonfat milk in TBST, probed using antibodies to Dec1, p21Cip1 , p16Ink4a , p15Ink4b , glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all from Santa Cruz Biotechnology, Santa Cruz, CA), p53 (Novocastra), and phospho-p53 (Cell Signaling), and detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Santa Cruz, CA) using ECL detection reagent (Roche).

    Techniques: Expressing, Immunostaining, Mouse Assay, Staining

    WRN depletion activates a p53 response in MSI cells. a, Phospho-p53 (S15) IF following sgRNA transduction in ovarian cell lines (50 µm scale bar). b, Nuclear phospho-p53 (S15) staining intensity per cell following WRN knockout compared to control sgRNA. Mean log fold-change: 0.059 (OVK18), −0.037 (ES2). Difference in log fold-change between OVK18 and ES2; P value (contrast test of least-squares means)

    Journal: Nature

    Article Title: WRN Helicase is a Synthetic Lethal Target in Microsatellite Unstable Cancers

    doi: 10.1038/s41586-019-1102-x

    Figure Lengend Snippet: WRN depletion activates a p53 response in MSI cells. a, Phospho-p53 (S15) IF following sgRNA transduction in ovarian cell lines (50 µm scale bar). b, Nuclear phospho-p53 (S15) staining intensity per cell following WRN knockout compared to control sgRNA. Mean log fold-change: 0.059 (OVK18), −0.037 (ES2). Difference in log fold-change between OVK18 and ES2; P value (contrast test of least-squares means)

    Article Snippet: Types of primary antibodies and the dilutions used for immunofluorescence were as follows: anti-ɣH2AX (Millipore Sigma, 05–636, 1:400); anti-p21 (Santa Cruz Biotechnology, sc-6246, 1:100); anti-phospho ATM [S1981] (Millipore Sigma, 05–740, 1:200); anti-phospho Chk2 [T68] (R & D Systems, AF1626, 1:100); anti-fibrillarin (Abcam, ab5821, 1:500); anti-phospho p53 [S15] (Cell Signaling Technology, 9284, 1:100); anti-WRN (Sigma, W0393, 1:200).

    Techniques: Transduction, Staining, Knock-Out

    WRN depletion in MSI cells induces cell cycle arrest, apoptosis, and a p53 response. a, GSEA enrichment/depletion scores in WRN-depleted OVK18 cells plotted against WRN-depleted SW48 cells. Signature enrichment plots for Hallmark gene sets shown for WRN-depleted OVK18 and SW48 cells. n = 2 biological replicates. b, phospho-p53 (S15) immunofluorescence (IF) following sgRNA transduction (50 µm scale bar). c, Nuclear phospho-p53 (S15) staining intensity per cell. Lower error bar, box lower limit, bar, box upper limit, upper error bar, dots: 1st, 25th percentiles, median, 75th, 99th percentiles, outliers, respectively. Mean log intensity change following WRN knockout compared to control sgRNA in MSI versus MSS cells; P

    Journal: Nature

    Article Title: WRN Helicase is a Synthetic Lethal Target in Microsatellite Unstable Cancers

    doi: 10.1038/s41586-019-1102-x

    Figure Lengend Snippet: WRN depletion in MSI cells induces cell cycle arrest, apoptosis, and a p53 response. a, GSEA enrichment/depletion scores in WRN-depleted OVK18 cells plotted against WRN-depleted SW48 cells. Signature enrichment plots for Hallmark gene sets shown for WRN-depleted OVK18 and SW48 cells. n = 2 biological replicates. b, phospho-p53 (S15) immunofluorescence (IF) following sgRNA transduction (50 µm scale bar). c, Nuclear phospho-p53 (S15) staining intensity per cell. Lower error bar, box lower limit, bar, box upper limit, upper error bar, dots: 1st, 25th percentiles, median, 75th, 99th percentiles, outliers, respectively. Mean log intensity change following WRN knockout compared to control sgRNA in MSI versus MSS cells; P

    Article Snippet: Types of primary antibodies and the dilutions used for immunofluorescence were as follows: anti-ɣH2AX (Millipore Sigma, 05–636, 1:400); anti-p21 (Santa Cruz Biotechnology, sc-6246, 1:100); anti-phospho ATM [S1981] (Millipore Sigma, 05–740, 1:200); anti-phospho Chk2 [T68] (R & D Systems, AF1626, 1:100); anti-fibrillarin (Abcam, ab5821, 1:500); anti-phospho p53 [S15] (Cell Signaling Technology, 9284, 1:100); anti-WRN (Sigma, W0393, 1:200).

    Techniques: Immunofluorescence, Transduction, Staining, Knock-Out

    Complex reduces proliferation of MCF7 cell line and enhances expression of p53 target genes in p53-dependent manner. a Complex negatively affects viability of MCF7 p53wt and MCF7 p53 –/– tumor cells. MCF7 p53wt (dark gray), MCF7 p53 −/− (light gray) cells were treated with Complex (50 μM), Ligand (50 μM and 100 μM), DMSO (1%, vehicle control), and doxorubicin (4 μM) for 24 h. Cell viability was evaluated by colorimetric MTS assay. Ligand demonstrated no cytotoxicity for either of the cell lines at both concentrations, whereas Complex substantially reduced viability that was comparable to doxorubicin at indicated concentration. Data are expressed as mean ± S.D., n = 3; * p

    Journal: Cell Death Discovery

    Article Title: Isatin-Schiff base-copper (II) complex induces cell death in p53-positive tumors

    doi: 10.1038/s41420-018-0120-z

    Figure Lengend Snippet: Complex reduces proliferation of MCF7 cell line and enhances expression of p53 target genes in p53-dependent manner. a Complex negatively affects viability of MCF7 p53wt and MCF7 p53 –/– tumor cells. MCF7 p53wt (dark gray), MCF7 p53 −/− (light gray) cells were treated with Complex (50 μM), Ligand (50 μM and 100 μM), DMSO (1%, vehicle control), and doxorubicin (4 μM) for 24 h. Cell viability was evaluated by colorimetric MTS assay. Ligand demonstrated no cytotoxicity for either of the cell lines at both concentrations, whereas Complex substantially reduced viability that was comparable to doxorubicin at indicated concentration. Data are expressed as mean ± S.D., n = 3; * p

    Article Snippet: Membranes were blocked with phosphate-buffered saline (PBS) with tween 20 containing 5% (mass/vol) nonfat dried milk for 1 h at RT, incubated with primary anti-p53 antibodies (Abcam, USA) overnight at 4 °C, and then with Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich, USA) for 1 h. THE Beta Actin Antibody [HRP] (GenScript, USA) was used for detection of Beta Actin as loading control.

    Techniques: Expressing, MTS Assay, Concentration Assay

    Complex induces apoptosis and necrosis in MCF7 p53wt and MCF7 p53 –/– tumor cells. a MCF7 p53wt and b MCF7 p53 −/− cells were treated with Complex (50 μM), Ligand (50 μM), and DMSO (1%, vehicle control) for 48 h, then stained with APC Annexin V and propidium iodide to evaluate percentage of apoptotic and necrotic cells. Stained cells were analyzed by flow cytometry using BD FACSAria III and data processed with FlowJo software package. Quadrants represent following: Q1—early apoptotic cells (Annexin V positive); Q2—late apoptotic and necrotic cells (double positive); Q3—healthy live cells (double negative); Q4—necrotic cells (PI positive). Diagrams show one representative dataset out of three independent experiments. Percentages are expressed as mean ± S.D., n = 3

    Journal: Cell Death Discovery

    Article Title: Isatin-Schiff base-copper (II) complex induces cell death in p53-positive tumors

    doi: 10.1038/s41420-018-0120-z

    Figure Lengend Snippet: Complex induces apoptosis and necrosis in MCF7 p53wt and MCF7 p53 –/– tumor cells. a MCF7 p53wt and b MCF7 p53 −/− cells were treated with Complex (50 μM), Ligand (50 μM), and DMSO (1%, vehicle control) for 48 h, then stained with APC Annexin V and propidium iodide to evaluate percentage of apoptotic and necrotic cells. Stained cells were analyzed by flow cytometry using BD FACSAria III and data processed with FlowJo software package. Quadrants represent following: Q1—early apoptotic cells (Annexin V positive); Q2—late apoptotic and necrotic cells (double positive); Q3—healthy live cells (double negative); Q4—necrotic cells (PI positive). Diagrams show one representative dataset out of three independent experiments. Percentages are expressed as mean ± S.D., n = 3

    Article Snippet: Membranes were blocked with phosphate-buffered saline (PBS) with tween 20 containing 5% (mass/vol) nonfat dried milk for 1 h at RT, incubated with primary anti-p53 antibodies (Abcam, USA) overnight at 4 °C, and then with Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich, USA) for 1 h. THE Beta Actin Antibody [HRP] (GenScript, USA) was used for detection of Beta Actin as loading control.

    Techniques: Staining, Flow Cytometry, Cytometry, Software

    Complex activates p53 and reduces proliferation of HCT116, MCF7 tumor cells. a Chemical structures of Isatin-Schiff base and its copper(II) complex. (E)-1-methyl-3-(phenylimino)indolin-2-one and (E)-1-methyl-3-(phenylimino)indolin-2-one copper(II) chloride complex are named as Ligand and Complex, respectively. b Complex, but not Ligand, activates p53 protein. Immunoblot analysis of MCF7 p53wt cells treated with Complex (50 µM), Ligand (50 µM), and DMSO (1%, vehicle control) for 24 h revealed Complex-mediated activation of p53 protein. c Lack of p53 protein expression in MCF7 p53 −/− validated by immunoblotting. MCF7 p53 −/− cells were obtained using CRISPR/Cas9 knockout of TP53 gene in MCF7 p53wt cells. Monoclonal populations of MCF7 cells containing frameshift in double-strand break region of TP53 gene were analyzed by immunoblotting to confirm lack of p53 protein expression. As a control we used mixed population of MCF7 cells stably transduced with lentivirus encoding pCW-Cas9 and transiently transfected with pLenti-SG1 construct encoding scrambled sgRNA. Both MCF7 p53wt and MCF7 p53 −/− cells were incubated with 1.5 µg/ml doxorubicin for 20 h prior collection and lysis. d , e Isatin-Schiff base-copper(II) complex negatively affects proliferation of HCT116 and MCF7 tumor cells. d HCT116 and e MCF7 cells were seeded at 5,000 per well in E-Plates 16 and were treated with Ligand (50 and 100 μM), Complex (50 μM), CuCl 2 (50 μM), and doxorubicin (4 μM) for 72 h. Treatments were performed in triplicates within each plate. Cell index parameter was recorded every 15 min

    Journal: Cell Death Discovery

    Article Title: Isatin-Schiff base-copper (II) complex induces cell death in p53-positive tumors

    doi: 10.1038/s41420-018-0120-z

    Figure Lengend Snippet: Complex activates p53 and reduces proliferation of HCT116, MCF7 tumor cells. a Chemical structures of Isatin-Schiff base and its copper(II) complex. (E)-1-methyl-3-(phenylimino)indolin-2-one and (E)-1-methyl-3-(phenylimino)indolin-2-one copper(II) chloride complex are named as Ligand and Complex, respectively. b Complex, but not Ligand, activates p53 protein. Immunoblot analysis of MCF7 p53wt cells treated with Complex (50 µM), Ligand (50 µM), and DMSO (1%, vehicle control) for 24 h revealed Complex-mediated activation of p53 protein. c Lack of p53 protein expression in MCF7 p53 −/− validated by immunoblotting. MCF7 p53 −/− cells were obtained using CRISPR/Cas9 knockout of TP53 gene in MCF7 p53wt cells. Monoclonal populations of MCF7 cells containing frameshift in double-strand break region of TP53 gene were analyzed by immunoblotting to confirm lack of p53 protein expression. As a control we used mixed population of MCF7 cells stably transduced with lentivirus encoding pCW-Cas9 and transiently transfected with pLenti-SG1 construct encoding scrambled sgRNA. Both MCF7 p53wt and MCF7 p53 −/− cells were incubated with 1.5 µg/ml doxorubicin for 20 h prior collection and lysis. d , e Isatin-Schiff base-copper(II) complex negatively affects proliferation of HCT116 and MCF7 tumor cells. d HCT116 and e MCF7 cells were seeded at 5,000 per well in E-Plates 16 and were treated with Ligand (50 and 100 μM), Complex (50 μM), CuCl 2 (50 μM), and doxorubicin (4 μM) for 72 h. Treatments were performed in triplicates within each plate. Cell index parameter was recorded every 15 min

    Article Snippet: Membranes were blocked with phosphate-buffered saline (PBS) with tween 20 containing 5% (mass/vol) nonfat dried milk for 1 h at RT, incubated with primary anti-p53 antibodies (Abcam, USA) overnight at 4 °C, and then with Anti-Mouse IgG–Peroxidase antibody (Sigma-Aldrich, USA) for 1 h. THE Beta Actin Antibody [HRP] (GenScript, USA) was used for detection of Beta Actin as loading control.

    Techniques: Western Blot, Activation Assay, Expressing, CRISPR, Knock-Out, Stable Transfection, Transduction, Transfection, Construct, Incubation, Lysis

    PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO – for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO − or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity ( Woo et al., 2008 ). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: PKCζ–PIASy association is critical for p53 SUMOylation and p53–Bcl-2 binding. (A) HUVECs were stimulated with 100 µM ONOO – for the indicated times and subjected to immunoprecipitation with anti-PIASy followed by Western blotting with anti-PKCζ (top). (B and C) Association between PKCζ and PIASy was tested by a mammalian two-hybrid assay. HeLa cells were transfected with plasmids containing Gal4-PKCζ wild type and VP16-PIASy (B) or truncated mutants of VP16-PIASy (C) as well as the Gal4-responsive luciferase reporter pG5-luc. After 24 h of transfection, cells were stimulated with 100 µM ONOO − or vehicle for 16 h, and luciferase activity was quantified. Luciferase activity was normalized with the Renilla luciferase (Luc.) activity ( Woo et al., 2008 ). Data are representative of three experiments using two or more different preparations of ECs (means ± SD; **, P

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Binding Assay, Immunoprecipitation, Western Blot, Two Hybrid Assay, Transfection, Luciferase, Activity Assay

    D-flow–induced PKCζ–PIASy association in nuclei and p53–Bcl-2 binding in the cytosol. (A and B) HUVECs were stimulated with either static or d-flow for 3 h and immunoassayed with antibodies of mouse anti-PKCζ and rabbit anti-PIASy (A) or mouse anti-p53 and rabbit anti–Bcl-2 (B). After d-flow stimulation, yellow in the merged images represent colocalization between PKCζ and PIASy in nuclei or p53 and Bcl-2 in cytosol. Images were recorded using a confocal microscope equipped with a Plapon 60× 1.42 NA oil lens objective. Shown are representative images from cells analyzed from three independent experiments in which ≥30 cells were analyzed per experiment. Bars, 10 µm.

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: D-flow–induced PKCζ–PIASy association in nuclei and p53–Bcl-2 binding in the cytosol. (A and B) HUVECs were stimulated with either static or d-flow for 3 h and immunoassayed with antibodies of mouse anti-PKCζ and rabbit anti-PIASy (A) or mouse anti-p53 and rabbit anti–Bcl-2 (B). After d-flow stimulation, yellow in the merged images represent colocalization between PKCζ and PIASy in nuclei or p53 and Bcl-2 in cytosol. Images were recorded using a confocal microscope equipped with a Plapon 60× 1.42 NA oil lens objective. Shown are representative images from cells analyzed from three independent experiments in which ≥30 cells were analyzed per experiment. Bars, 10 µm.

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Flow Cytometry, Binding Assay, Microscopy

    D-flow induces p53 SUMOylation and apoptosis via PIASy activation. (A) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with d-flow for the indicated times. p53 SUMOylation, expression of PIASy, p53, and SUMO2/3 were detected as described in Material and methods. Densitometric analyses of p53 SUMOylation were performed as described in Fig. 1 . (B and C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with d-flow for 36 h, apoptotic nuclei were detected by TUNEL staining (B, bottom), and Western blotting with anti–cleaved caspase 3 (C, top) was performed. Immunoblots of PIASy conformed depletion of PIASy by the specific siRNA (B, top). Densitometry analysis of cleaved caspase 3 expression was performed as described in Fig. 2 C (bottom). The experiments were performed in triplicate using three different batches of d-flow–stimulated HUVECs. (D) HUVECs were transduced with an adenovirus vector containing p53, p53-K386R (KR; sumoylation defect mutant), or p53-ΔNES (L348,350A; NES mutant) for 24 h and then stimulated with d-flow for 36 h followed by TUNEL staining as described in Materials and methods. (E, top) Quantification of apoptosis shown as the percentage of TUNEL-positive cells. Bars, 30 µm. (bottom) Equal expressions of p53, p53-K386R, and p53-ΔNES were analyzed by Western blotting in ECs. Data are from three separate experiments using two or more different EC preparations. Error bars show means ± SD; *, P

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: D-flow induces p53 SUMOylation and apoptosis via PIASy activation. (A) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with d-flow for the indicated times. p53 SUMOylation, expression of PIASy, p53, and SUMO2/3 were detected as described in Material and methods. Densitometric analyses of p53 SUMOylation were performed as described in Fig. 1 . (B and C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with d-flow for 36 h, apoptotic nuclei were detected by TUNEL staining (B, bottom), and Western blotting with anti–cleaved caspase 3 (C, top) was performed. Immunoblots of PIASy conformed depletion of PIASy by the specific siRNA (B, top). Densitometry analysis of cleaved caspase 3 expression was performed as described in Fig. 2 C (bottom). The experiments were performed in triplicate using three different batches of d-flow–stimulated HUVECs. (D) HUVECs were transduced with an adenovirus vector containing p53, p53-K386R (KR; sumoylation defect mutant), or p53-ΔNES (L348,350A; NES mutant) for 24 h and then stimulated with d-flow for 36 h followed by TUNEL staining as described in Materials and methods. (E, top) Quantification of apoptosis shown as the percentage of TUNEL-positive cells. Bars, 30 µm. (bottom) Equal expressions of p53, p53-K386R, and p53-ΔNES were analyzed by Western blotting in ECs. Data are from three separate experiments using two or more different EC preparations. Error bars show means ± SD; *, P

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Flow Cytometry, Activation Assay, Transfection, Expressing, TUNEL Assay, Staining, Western Blot, Transduction, Plasmid Preparation, Mutagenesis

    ONOO − mediates d-flow–induced PKCζ activation, p53 SUMOylation, and EC apoptosis. (A) ONOO − mediates d-flow–induced PKCζ activation and p53 SUMOylation. HUVECs were pretreated by 5 µM ebselen, 20 µM L-NAME, and 10 µM Mn-TBAP for 30 min and exposed to d-flow for 3 h. PKCζ phosphorylation at Thr560 and p53 SUMOylation were determined as described in Materials and methods. (B and C) Densitometry analyses of p53 SUMOylation (B) and PKCζ phosphorylation (C) were performed as described in Fig. 1 . **, P

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: ONOO − mediates d-flow–induced PKCζ activation, p53 SUMOylation, and EC apoptosis. (A) ONOO − mediates d-flow–induced PKCζ activation and p53 SUMOylation. HUVECs were pretreated by 5 µM ebselen, 20 µM L-NAME, and 10 µM Mn-TBAP for 30 min and exposed to d-flow for 3 h. PKCζ phosphorylation at Thr560 and p53 SUMOylation were determined as described in Materials and methods. (B and C) Densitometry analyses of p53 SUMOylation (B) and PKCζ phosphorylation (C) were performed as described in Fig. 1 . **, P

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Flow Cytometry, Activation Assay

    PKCζ mediates ONOO − -induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; Kern et al., 1992 ). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO − for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: PKCζ mediates ONOO − -induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; Kern et al., 1992 ). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO − for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Binding Assay, Activity Assay, Transfection, Luciferase, Plasmid Preparation, Sequencing

    PKCζ mediates d-flow–induced p53 SUMOylation and p53–Bcl-2 binding. (A) HeLa cells were transfected for 24 h as indicated with Flag-tagged p53, HA-tagged SUMO3, and HA-tagged CATζ. p53 SUMOylation was detected by immunoprecipitation with anti-Flag followed by Western blotting with anti-SUMO2/3 (top). Both protein expression and immunoprecipitated p53 were confirmed by anti-Flag antibody, and CATζ and SUMO expression were detected with anti-HA. Mono-SUMOylation band (∼74 kD) and poly-SUMOylation bands ( > 78 kD) were detected. The asterisk indicates mono-SUMOylation of p52. (B) HUVECs were transfected for 24 h with either PIASy or control siRNA as indicated, and then the cells were transfected with HA-CATζ or vector alone for another 24 h. (top) p53 SUMOylation was detected by immunoprecipitation with anti-p53 followed by Western blotting with anti-SUMO2/3. PIASy expression was confirmed by immunoblotting with anti-PIASy, and p53, HA-CATζ, and SUMO expression was confirmed with anti-p53, -HA, and -SUMO2/3, respectively. (C) HUVECs were transfected with either p53 or control siRNA as indicated for 24 h, and then the cells were transduced with an Ad-SENP2 or LacZ with a control for another 24 h. p53 SUMOylation, expression of p53, SENP2, and SUMO were determined as described in Materials and methods. The asterisks indicate nonspecific bands. (D) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h and then stimulated with d-flow for the indicated times. p53 SUMOylation and p53–Bcl-2 binding were determined as described in Materials and methods. (left graph) Intensities of SUMOylated p53 bands at 74, 82, 130, and 185 kD were quantified by densitometry after subtracting background gel density. After normalization of each control as described in Fig. 1 , results were expressed relative to the SUMOylation level in static condition (0 min; 100%). Shown are means ± SD ( n = 3). **, P

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: PKCζ mediates d-flow–induced p53 SUMOylation and p53–Bcl-2 binding. (A) HeLa cells were transfected for 24 h as indicated with Flag-tagged p53, HA-tagged SUMO3, and HA-tagged CATζ. p53 SUMOylation was detected by immunoprecipitation with anti-Flag followed by Western blotting with anti-SUMO2/3 (top). Both protein expression and immunoprecipitated p53 were confirmed by anti-Flag antibody, and CATζ and SUMO expression were detected with anti-HA. Mono-SUMOylation band (∼74 kD) and poly-SUMOylation bands ( > 78 kD) were detected. The asterisk indicates mono-SUMOylation of p52. (B) HUVECs were transfected for 24 h with either PIASy or control siRNA as indicated, and then the cells were transfected with HA-CATζ or vector alone for another 24 h. (top) p53 SUMOylation was detected by immunoprecipitation with anti-p53 followed by Western blotting with anti-SUMO2/3. PIASy expression was confirmed by immunoblotting with anti-PIASy, and p53, HA-CATζ, and SUMO expression was confirmed with anti-p53, -HA, and -SUMO2/3, respectively. (C) HUVECs were transfected with either p53 or control siRNA as indicated for 24 h, and then the cells were transduced with an Ad-SENP2 or LacZ with a control for another 24 h. p53 SUMOylation, expression of p53, SENP2, and SUMO were determined as described in Materials and methods. The asterisks indicate nonspecific bands. (D) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h and then stimulated with d-flow for the indicated times. p53 SUMOylation and p53–Bcl-2 binding were determined as described in Materials and methods. (left graph) Intensities of SUMOylated p53 bands at 74, 82, 130, and 185 kD were quantified by densitometry after subtracting background gel density. After normalization of each control as described in Fig. 1 , results were expressed relative to the SUMOylation level in static condition (0 min; 100%). Shown are means ± SD ( n = 3). **, P

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Flow Cytometry, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Transduction

    Increases in phosphorylated and total PKCζ and nonnuclear p53 expression within the d-flow regions (HP areas) and decreased apoptosis in ECs of p53 −/− mice. (A) A representative epifluorescence image of the whole specimen. Fixed aortas of wild-type mice were cut longitudinally, and the arch region was further cut into two halves. Areas of d-flow (HP area; lesser curvature) are outlined in red, and neighboring areas of s-flow (LP area) are lined in blue. a, artery. (B and C) En face preparations were double stained with anti–VE-cadherin (VE-cad; used as an EC marker) and an anti–total PKCζ antibody (B) or phospho-PKCζ T560 antibody (C). X-y axis images were collected at 0.5-µm increments so that a z stack of ∼4-µm thickness from the luminal surface was obtained. From each image background, fluorescence intensity was subtracted, and the pixel number of the stained region per unit area of the endothelium in HP and LP area within the aortic arch was determined ( n = 3). Areas of d-flow (HP areas; lesser curvature) show both increased total and phospho-PKCζ expression compared with the neighboring areas of s-flow (LP area). Bars, 20 µm. Bar graphs show quantification of total (B) and phospho (C)-PKCζ in HP and LP areas. Data are shown as means ± SEM; *, P

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: Increases in phosphorylated and total PKCζ and nonnuclear p53 expression within the d-flow regions (HP areas) and decreased apoptosis in ECs of p53 −/− mice. (A) A representative epifluorescence image of the whole specimen. Fixed aortas of wild-type mice were cut longitudinally, and the arch region was further cut into two halves. Areas of d-flow (HP area; lesser curvature) are outlined in red, and neighboring areas of s-flow (LP area) are lined in blue. a, artery. (B and C) En face preparations were double stained with anti–VE-cadherin (VE-cad; used as an EC marker) and an anti–total PKCζ antibody (B) or phospho-PKCζ T560 antibody (C). X-y axis images were collected at 0.5-µm increments so that a z stack of ∼4-µm thickness from the luminal surface was obtained. From each image background, fluorescence intensity was subtracted, and the pixel number of the stained region per unit area of the endothelium in HP and LP area within the aortic arch was determined ( n = 3). Areas of d-flow (HP areas; lesser curvature) show both increased total and phospho-PKCζ expression compared with the neighboring areas of s-flow (LP area). Bars, 20 µm. Bar graphs show quantification of total (B) and phospho (C)-PKCζ in HP and LP areas. Data are shown as means ± SEM; *, P

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Expressing, Flow Cytometry, Mouse Assay, Staining, Marker, Fluorescence

    ONOO − induces p53 SUMOylation and p53–Bcl-2 binding via PIASy activation. (A and B) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with 100 µM ONOO – for the indicated times. p53 SUMOylation (A) and p53–Bcl-2 binding (B) were determined as described in Materials and methods. (left) PIASy and p53 expressions were detected by Western blotting with appropriate specific antibodies. Densitometric analyses of p53 SUMOylation (A) and p53–Bcl-2 binding (B) were performed as described in Fig. 1 . (C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with 100 µM ONOO − for 8 h, apoptotic nuclei were detected by TUNEL staining. Data are expressed as mean percentages ± SD from three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: PKC? mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation

    doi: 10.1083/jcb.201010051

    Figure Lengend Snippet: ONOO − induces p53 SUMOylation and p53–Bcl-2 binding via PIASy activation. (A and B) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with 100 µM ONOO – for the indicated times. p53 SUMOylation (A) and p53–Bcl-2 binding (B) were determined as described in Materials and methods. (left) PIASy and p53 expressions were detected by Western blotting with appropriate specific antibodies. Densitometric analyses of p53 SUMOylation (A) and p53–Bcl-2 binding (B) were performed as described in Fig. 1 . (C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with 100 µM ONOO − for 8 h, apoptotic nuclei were detected by TUNEL staining. Data are expressed as mean percentages ± SD from three independent experiments. *, P

    Article Snippet: Antibodies, siRNA, adenovirus, and reagents Rabbit and mouse anti-PKCζ (C-20 [SC-216] and A-3 [SC-17781]), rabbit and mouse anti-p53 (FL-393 [SC-6243] and DO-1 [SC-126]), rabbit and mouse anti–Bcl-2 (N-19 [SC-492] and C-2 [SC-7382]), rabbit and mouse anti-HA (Y-11 [SC-805] and F-7 [SC-7392]), and anti-myc (A-14; SC-789) were purchased from Santa Cruz Biotechnology, Inc.

    Techniques: Binding Assay, Activation Assay, Transfection, Western Blot, TUNEL Assay, Staining

    Mutant p53 (R280K) expression correlates with Nox4 expression and overrides wild-type p53 (WT-p53) repression of Nox4 in MCF-10A breast epithelial cells. ( A ) MCF-10A cells were transfected with empty vector control, p53-WT, p53-R175H, or p53-R280K plasmids for 24 h followed by treatment with or without TGF- β (10 ng ml −1 ) for an additional 24 h. Nox4- and GAPDH-specific primers were used for real-time quantitative PCR amplification of total cDNA reverse transcribed from cells. Results are described as relative quantification of Nox4 mRNA relative to vector untreated control ( n =3). ( B ) Detection of Nox4 protein by western blotting. MCF-10A cells were transfected with non-targeting control (50 n M ) or SMARTpool Nox4-specific siRNAs (50 n M ) for 72 h, and either left untreated or treated with TGF- β (5 ng ml −1 ) for an additional 24 h (left panel). Control blots (right panels) detect a transfected Nox4 cDNA product of the same size. Immunoblots were probed with anti-Nox4 followed by anti-GAPDH antibodies. ( C ) MCF-10A cells were transfected with control siRNAs (50 n M ) or p53-specific siRNAs (50 n M ) for 72 h followed by treatment with or without TGF- β (10 ng ml −1 ) for 24 h. Protein expression was analysed by immunoblotting 40 μ g of total cell lysate and sequentially probed with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( D ) MCF-10A cells were treated with 10 or 20 μ M Nutlin-3 for 24 h. Protein expression was analysed by immunoblotting 40 μ g of total cell lysate and sequentially probed with antibodies against Nox4, p53, and GAPDH sequentially. ( E ) MCF-10A cells were co-transfected with vector alone or Nox4-DN and p53-WT, or p53-R280K plasmids. Twenty-four hours post transfection, cells were re-seeded in the upper chamber of a Matrigel transwell and incubated with the lower chamber containing DMEM/F12 medium with TGF- β 10 ng ml −1 . After 24 h, the migrating cells were fixed, stained, and counted from 10 random fields ( n =3). ( F ) MCF-10A cells were treated with transfection reagent alone or transfected with control or p53-specific siRNAs. After 72 h, the cells were re-seeded in the upper chamber of a Matrigel transwell and incubated in the lower chamber containing DMEM/F12 medium with TGF- β 5 ng ml −1 for 24 h. The migrating cells were counted from 10 random fields ( n =2 in triplicate).

    Journal: British Journal of Cancer

    Article Title: Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    doi: 10.1038/bjc.2014.165

    Figure Lengend Snippet: Mutant p53 (R280K) expression correlates with Nox4 expression and overrides wild-type p53 (WT-p53) repression of Nox4 in MCF-10A breast epithelial cells. ( A ) MCF-10A cells were transfected with empty vector control, p53-WT, p53-R175H, or p53-R280K plasmids for 24 h followed by treatment with or without TGF- β (10 ng ml −1 ) for an additional 24 h. Nox4- and GAPDH-specific primers were used for real-time quantitative PCR amplification of total cDNA reverse transcribed from cells. Results are described as relative quantification of Nox4 mRNA relative to vector untreated control ( n =3). ( B ) Detection of Nox4 protein by western blotting. MCF-10A cells were transfected with non-targeting control (50 n M ) or SMARTpool Nox4-specific siRNAs (50 n M ) for 72 h, and either left untreated or treated with TGF- β (5 ng ml −1 ) for an additional 24 h (left panel). Control blots (right panels) detect a transfected Nox4 cDNA product of the same size. Immunoblots were probed with anti-Nox4 followed by anti-GAPDH antibodies. ( C ) MCF-10A cells were transfected with control siRNAs (50 n M ) or p53-specific siRNAs (50 n M ) for 72 h followed by treatment with or without TGF- β (10 ng ml −1 ) for 24 h. Protein expression was analysed by immunoblotting 40 μ g of total cell lysate and sequentially probed with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( D ) MCF-10A cells were treated with 10 or 20 μ M Nutlin-3 for 24 h. Protein expression was analysed by immunoblotting 40 μ g of total cell lysate and sequentially probed with antibodies against Nox4, p53, and GAPDH sequentially. ( E ) MCF-10A cells were co-transfected with vector alone or Nox4-DN and p53-WT, or p53-R280K plasmids. Twenty-four hours post transfection, cells were re-seeded in the upper chamber of a Matrigel transwell and incubated with the lower chamber containing DMEM/F12 medium with TGF- β 10 ng ml −1 . After 24 h, the migrating cells were fixed, stained, and counted from 10 random fields ( n =3). ( F ) MCF-10A cells were treated with transfection reagent alone or transfected with control or p53-specific siRNAs. After 72 h, the cells were re-seeded in the upper chamber of a Matrigel transwell and incubated in the lower chamber containing DMEM/F12 medium with TGF- β 5 ng ml −1 for 24 h. The migrating cells were counted from 10 random fields ( n =2 in triplicate).

    Article Snippet: Antibodies and immunoblotting analysis Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.

    Techniques: Mutagenesis, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Amplification, Western Blot, Incubation, Staining

    Exogenous expression of mutant p53 (R280K) induces Nox4 in confluent and motile MCF-10A cells. MCF-10A cells were transfected with p53-R280K for 48 h. Fluorescence microscopy images were taken from the edge of a sub-confluent layer of cells (upper row) and from a confluent monolayer (lower row). From left to right, cells were stained with anti-p53 antibodies detecting high expression of transfected p53-R280K (left panel), endogenous Nox4 protein expression was detected with anti-Nox4 antibodies (middle panel), and DAPI staining of nuclei, (right panel). Dotted white line in the upper panels indicates the edge of a monolayer of cells in a sub-confluent well of a chambered coverglass.

    Journal: British Journal of Cancer

    Article Title: Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    doi: 10.1038/bjc.2014.165

    Figure Lengend Snippet: Exogenous expression of mutant p53 (R280K) induces Nox4 in confluent and motile MCF-10A cells. MCF-10A cells were transfected with p53-R280K for 48 h. Fluorescence microscopy images were taken from the edge of a sub-confluent layer of cells (upper row) and from a confluent monolayer (lower row). From left to right, cells were stained with anti-p53 antibodies detecting high expression of transfected p53-R280K (left panel), endogenous Nox4 protein expression was detected with anti-Nox4 antibodies (middle panel), and DAPI staining of nuclei, (right panel). Dotted white line in the upper panels indicates the edge of a monolayer of cells in a sub-confluent well of a chambered coverglass.

    Article Snippet: Antibodies and immunoblotting analysis Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.

    Techniques: Expressing, Mutagenesis, Transfection, Fluorescence, Microscopy, Staining

    Mutant p53 proteins support TGF- β -induced Nox4 and cell migration. ( A ) H1299 cells were transfected with vector control plasmid, wild-type p53 (WT-p53), p53-R175H mutant, or p53-R280K mutant plasmids. Twenty-four hours post transfection, the cells were treated with TGF- β (5 ng ml −1 ) or left untreated for an additional 24 h. Nox4-specific primers were used for quantitative real-time PCR. Nox4- and GAPDH-specific primers were used for quantitative PCR ( n =3). Results are described as relative quantification of Nox4 mRNA relative to vector untreated control. ( B ) Nox4 protein is differentially regulated by WT and mutant p53 expression. H1299 cells were transfected and treated as described in A . Forty micrograms of total cell lysate were analysed by western blotting. The blot was probed sequentially with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( C ) H1299 cells were transfected with vector alone, p53-WT, p53-R175H, or p53-R280K, and co-transfected with either vector control or dominant-negative Nox4 (Nox4-DN) plasmids. Twenty-four hours after transfection, cells were treated with TGF- β (5 ng ml −1 ) for 24 h. Cells were then collected and assayed for superoxide generation ( n =3, in triplicate). ( D ) H1299 cells were transfected as in C for 24 h. The cells were then re-seeded in the upper chamber of a Matrigel transwell and incubated in the lower chamber containing RPMI-1640 medium containing TGF- β 5 ng ml −1 . After 24 h, the migrating cells were fixed, stained, and counted from 10 random fields ( n =3). ( E , F ) H1299 cells were transfected with vector control, p53-WT, or p53-R280K. After 24 h, the cells were treated with 616451 (10 μ M ), a TGF- β receptor I-specific inhibitor or SIS3 (10 μ M ), a SMAD3-specific inhibitor for 4 h before treating with TGF- β for 20 h. Fibronectin-( E ) or Nox4 ( F )-specific primers were used for real-time quantitative PCR amplification of total cDNA reverse transcribed from cells. Results are described as relative quantification relative to vector untreated control. GAPDH-specific primers were used as an internal control ( n =3, in triplicate). Significant values are indicated as * P -value

    Journal: British Journal of Cancer

    Article Title: Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    doi: 10.1038/bjc.2014.165

    Figure Lengend Snippet: Mutant p53 proteins support TGF- β -induced Nox4 and cell migration. ( A ) H1299 cells were transfected with vector control plasmid, wild-type p53 (WT-p53), p53-R175H mutant, or p53-R280K mutant plasmids. Twenty-four hours post transfection, the cells were treated with TGF- β (5 ng ml −1 ) or left untreated for an additional 24 h. Nox4-specific primers were used for quantitative real-time PCR. Nox4- and GAPDH-specific primers were used for quantitative PCR ( n =3). Results are described as relative quantification of Nox4 mRNA relative to vector untreated control. ( B ) Nox4 protein is differentially regulated by WT and mutant p53 expression. H1299 cells were transfected and treated as described in A . Forty micrograms of total cell lysate were analysed by western blotting. The blot was probed sequentially with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( C ) H1299 cells were transfected with vector alone, p53-WT, p53-R175H, or p53-R280K, and co-transfected with either vector control or dominant-negative Nox4 (Nox4-DN) plasmids. Twenty-four hours after transfection, cells were treated with TGF- β (5 ng ml −1 ) for 24 h. Cells were then collected and assayed for superoxide generation ( n =3, in triplicate). ( D ) H1299 cells were transfected as in C for 24 h. The cells were then re-seeded in the upper chamber of a Matrigel transwell and incubated in the lower chamber containing RPMI-1640 medium containing TGF- β 5 ng ml −1 . After 24 h, the migrating cells were fixed, stained, and counted from 10 random fields ( n =3). ( E , F ) H1299 cells were transfected with vector control, p53-WT, or p53-R280K. After 24 h, the cells were treated with 616451 (10 μ M ), a TGF- β receptor I-specific inhibitor or SIS3 (10 μ M ), a SMAD3-specific inhibitor for 4 h before treating with TGF- β for 20 h. Fibronectin-( E ) or Nox4 ( F )-specific primers were used for real-time quantitative PCR amplification of total cDNA reverse transcribed from cells. Results are described as relative quantification relative to vector untreated control. GAPDH-specific primers were used as an internal control ( n =3, in triplicate). Significant values are indicated as * P -value

    Article Snippet: Antibodies and immunoblotting analysis Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.

    Techniques: Mutagenesis, Migration, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Dominant Negative Mutation, Incubation, Staining, Amplification

    Wild-type p53 (WT-p53) and mutant p53 differentially regulate Nox4 expression in both TGF- β -dependent and independent mechanisms. Wild-type p53 suppresses basal Nox4 and TGF- β -induced Nox4 expression, whereas mutant p53 (R175H and R280K) positively regulates or enhances the TGF- β /SMAD3 effect on Nox4. Expression of mutant p53 alone can upregulate Nox4 mRNA and protein expression. The downstream effects of Nox4-dependent ROS contributes to increased fibronectin mRNA, phosphorylation and activation of FAK, and subsequent cell migration and invasion. This pathway can be inhibited with specific chemical inhibitors to TGF- β receptor-1 and SIS3, or overexpression of Nox4-DN can diminish TGF- β -induction of these events. Moreover, depletion of endogenous WT-p53 by siRNA increases Nox4 expression, whereas depleting endogenous mutant p53-R280K reduces Nox4 expression. Collectively, Nox4 is a mediator of pro-migratory events downstream of TGF- β and mutant p53, and thereby acts as an attractive target for managing or suppressing metastatic disease.

    Journal: British Journal of Cancer

    Article Title: Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    doi: 10.1038/bjc.2014.165

    Figure Lengend Snippet: Wild-type p53 (WT-p53) and mutant p53 differentially regulate Nox4 expression in both TGF- β -dependent and independent mechanisms. Wild-type p53 suppresses basal Nox4 and TGF- β -induced Nox4 expression, whereas mutant p53 (R175H and R280K) positively regulates or enhances the TGF- β /SMAD3 effect on Nox4. Expression of mutant p53 alone can upregulate Nox4 mRNA and protein expression. The downstream effects of Nox4-dependent ROS contributes to increased fibronectin mRNA, phosphorylation and activation of FAK, and subsequent cell migration and invasion. This pathway can be inhibited with specific chemical inhibitors to TGF- β receptor-1 and SIS3, or overexpression of Nox4-DN can diminish TGF- β -induction of these events. Moreover, depletion of endogenous WT-p53 by siRNA increases Nox4 expression, whereas depleting endogenous mutant p53-R280K reduces Nox4 expression. Collectively, Nox4 is a mediator of pro-migratory events downstream of TGF- β and mutant p53, and thereby acts as an attractive target for managing or suppressing metastatic disease.

    Article Snippet: Antibodies and immunoblotting analysis Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.

    Techniques: Mutagenesis, Expressing, Activation Assay, Migration, Over Expression

    Endogenous mutant p53 (R280K) supports TGF- β /SMAD-dependent Nox4 induction in MDA-MB-231 breast epithelial cells. ( A ) MDA-MB-231 cells were transfected with control siRNAs (50 n M ) or p53-specific siRNAs (50 n M ) for 72 h then simulated with TGF- β (5 ng ml −1 ) for 24 h. Real-time quantitative PCR analysis of Nox4 mRNA expression was determined from MDA-MB-231 cells treated as in A ( n =3, in triplicate). ( B ) MDA-MB-231 cells were treated as in A followed by protein expression analysis by immunoblotting 40 μ g of total cell lysate. The blot was sequentially probed with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( C ) MDA-MB-231 cells were transfected with non-targeting control (50 n M ) or SMARTpool Nox4-specific siRNAs (50 n M ) for 72 h and either left untreated or treated with TGF- β (5 ng ml −1 ) for an additional 24 h. Nox4 protein expression was analysed by western blotting. Immunoblots were probed with anti-Nox4 followed by anti-GAPDH. ( D ) MDA-MB-231 cells were treated as described and assayed for superoxide production with superoxide-specific Diogenes reagent for 1 h ( n =3, in triplicate). ( E ) MDA-MB-231 cells were treated with transfection reagent alone or transfected with control or p53-specific siRNAs. After 72 h, the cells were re-seeded in the upper chamber of a Matrigel transwell and incubated in the lower chamber containing RPMI-1640 medium containing TGF- β 5 ng ml −1 for 24 h. The migrating cells were counted from 10 random fields ( n =3). ( F ) MDA-MB-231 cells were left untreated or treated with DMSO (vehicle), 616451 (10 μ M ), or SIS3 (10 μ M ) for 4 h before the addition of TGF- β (5 ng ml −1 ) for 24 h. Protein expression was analysed by immunoblotting 40 μ g of total cell lysate and sequentially probed with the indicated antibodies. ( G ) Total RNA extracted from cells treated as in F was reverse transcribed for real-time quantitative PCR analysis of Nox4 mRNA expression. Results are described as relative quantification of Nox4 mRNA relative to untreated control ( n =3, in triplicate). Significant values are indicated as * P -value

    Journal: British Journal of Cancer

    Article Title: Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    doi: 10.1038/bjc.2014.165

    Figure Lengend Snippet: Endogenous mutant p53 (R280K) supports TGF- β /SMAD-dependent Nox4 induction in MDA-MB-231 breast epithelial cells. ( A ) MDA-MB-231 cells were transfected with control siRNAs (50 n M ) or p53-specific siRNAs (50 n M ) for 72 h then simulated with TGF- β (5 ng ml −1 ) for 24 h. Real-time quantitative PCR analysis of Nox4 mRNA expression was determined from MDA-MB-231 cells treated as in A ( n =3, in triplicate). ( B ) MDA-MB-231 cells were treated as in A followed by protein expression analysis by immunoblotting 40 μ g of total cell lysate. The blot was sequentially probed with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( C ) MDA-MB-231 cells were transfected with non-targeting control (50 n M ) or SMARTpool Nox4-specific siRNAs (50 n M ) for 72 h and either left untreated or treated with TGF- β (5 ng ml −1 ) for an additional 24 h. Nox4 protein expression was analysed by western blotting. Immunoblots were probed with anti-Nox4 followed by anti-GAPDH. ( D ) MDA-MB-231 cells were treated as described and assayed for superoxide production with superoxide-specific Diogenes reagent for 1 h ( n =3, in triplicate). ( E ) MDA-MB-231 cells were treated with transfection reagent alone or transfected with control or p53-specific siRNAs. After 72 h, the cells were re-seeded in the upper chamber of a Matrigel transwell and incubated in the lower chamber containing RPMI-1640 medium containing TGF- β 5 ng ml −1 for 24 h. The migrating cells were counted from 10 random fields ( n =3). ( F ) MDA-MB-231 cells were left untreated or treated with DMSO (vehicle), 616451 (10 μ M ), or SIS3 (10 μ M ) for 4 h before the addition of TGF- β (5 ng ml −1 ) for 24 h. Protein expression was analysed by immunoblotting 40 μ g of total cell lysate and sequentially probed with the indicated antibodies. ( G ) Total RNA extracted from cells treated as in F was reverse transcribed for real-time quantitative PCR analysis of Nox4 mRNA expression. Results are described as relative quantification of Nox4 mRNA relative to untreated control ( n =3, in triplicate). Significant values are indicated as * P -value

    Article Snippet: Antibodies and immunoblotting analysis Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.

    Techniques: Mutagenesis, Multiple Displacement Amplification, Transfection, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Incubation

    Wild-type p53 (WT-p53) suppresses TGF- β -induced Nox4 in p53-null H1299 lung epithelial cells. ( A ) H1299 cells were transfected with vector alone or WT-p53 cDNA. Twenty-four hours after transfection, cells were treated with TGF- β (5 ng ml −1 ) for 24 h. Human Nox4- and GAPDH-specific primers were used for PCR amplification of total cDNA reverse transcribed from cells ( n =3). Results are described as relative quantification of Nox4 mRNA relative to vector untreated expression using GAPDH as an internal control for normalisation. Inset shows immunoblot analysis of p53 protein expression in H1299 cells transfected with vector alone or p53-WT plasmids. ( B ) Nox4 protein is downregulated by p53-WT expression. H1299 cells were transfected and treated as described in A . Forty micrograms of total cell lysate were analysed by western blotting. The immunoblot was probed sequentially with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( C ) H1299 cells were transfected with non-targeting control or SMARTpool Nox4-specific siRNAs (50 n M ) for 72 h, and either left untreated or treated with TGF- β (5 ng ml −1 ) for an additional 24 h. Nox4 protein expression was analysed by western blotting. Immunoblots were probed with anti-Nox4 followed by anti-GAPDH antibodies. ( D ) H1299 cells were transfected with vector alone or p53-WT or co-transfected with dominant-negative Nox4 (Nox4-DN) cDNA. Twenty-four hours after transfection, cells were treated with TGF- β (5 ng ml −1 ) for 24 h. Cells were collected and assayed for superoxide production with superoxide-specific Diogenes reagent for 1 h ( n =3, in triplicate). ( E ) H1299 cells were transfected with either vector control or p53-WT plasmids and treated with TGF- β as in D and collected and assayed for H 2 O 2 production with luminol/HRP ( n =3, in triplicate). Significance values are indicated as * P -value

    Journal: British Journal of Cancer

    Article Title: Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    doi: 10.1038/bjc.2014.165

    Figure Lengend Snippet: Wild-type p53 (WT-p53) suppresses TGF- β -induced Nox4 in p53-null H1299 lung epithelial cells. ( A ) H1299 cells were transfected with vector alone or WT-p53 cDNA. Twenty-four hours after transfection, cells were treated with TGF- β (5 ng ml −1 ) for 24 h. Human Nox4- and GAPDH-specific primers were used for PCR amplification of total cDNA reverse transcribed from cells ( n =3). Results are described as relative quantification of Nox4 mRNA relative to vector untreated expression using GAPDH as an internal control for normalisation. Inset shows immunoblot analysis of p53 protein expression in H1299 cells transfected with vector alone or p53-WT plasmids. ( B ) Nox4 protein is downregulated by p53-WT expression. H1299 cells were transfected and treated as described in A . Forty micrograms of total cell lysate were analysed by western blotting. The immunoblot was probed sequentially with antibodies against Nox4, p53, phospho-SMAD3, and total SMAD3. ( C ) H1299 cells were transfected with non-targeting control or SMARTpool Nox4-specific siRNAs (50 n M ) for 72 h, and either left untreated or treated with TGF- β (5 ng ml −1 ) for an additional 24 h. Nox4 protein expression was analysed by western blotting. Immunoblots were probed with anti-Nox4 followed by anti-GAPDH antibodies. ( D ) H1299 cells were transfected with vector alone or p53-WT or co-transfected with dominant-negative Nox4 (Nox4-DN) cDNA. Twenty-four hours after transfection, cells were treated with TGF- β (5 ng ml −1 ) for 24 h. Cells were collected and assayed for superoxide production with superoxide-specific Diogenes reagent for 1 h ( n =3, in triplicate). ( E ) H1299 cells were transfected with either vector control or p53-WT plasmids and treated with TGF- β as in D and collected and assayed for H 2 O 2 production with luminol/HRP ( n =3, in triplicate). Significance values are indicated as * P -value

    Article Snippet: Antibodies and immunoblotting analysis Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.

    Techniques: Transfection, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Expressing, Western Blot, Dominant Negative Mutation

    Nox4 and mutant p53 (R280K) modulate TGF- β -induced activation of focal adhesion kinase (pFAK). ( A ) Overexpression of Nox4-DN reduces FAK phosphorylation. MDA-MB-231 cells were transfected with either vector control or V5-tagged Nox4-DN for 24 h followed by TGF- β (5 ng ml −1 ) stimulation for an additional 24 h. Total cell lysates collected for immunoblot analysis using antibodies against Nox4, p53, phospho-FAK (pFAK) and total FAK, and V5. ( B ) MDA-MB-231 cells were transfected with control siRNAs (25 n M ) or p53-specific siRNAs (25 n M ) for 72 h then simulated with TGF- β (5 ng ml −1 ) for 24 h. Protein expression was analysed by immunoblotting total cell lysates with antibodies against Nox4, p53, phospho-FAK (pFAK), and total FAK. ( C ) MDA-MB-231 cells were transfected with Nox4-DN and GFP for 24 h followed by TGF- β treatment for an additional 24 h. Upper panel: GFP (Nox4-DN) cells vs phospho-FAK staining. Lower panel: cells expressing Nox4-DN (arrows) have reduced phospho-FAK staining compared with surrounding non-transfected cells. ( D ) MCF-10A cells were co-transfected with vector alone or Nox4-DN, and p53-WT or p53-R280K plasmids then treated with TGF- β for 24 h. Immunoblotting analysis indicates phospho-FAK, FAK, V5-tagged Nox4-DN, and p53 protein expression. ( E ) p53-null H1299 cells were co-transfected transfected, treated, and analysed for protein expression and FAK phosphorylation as in D . ( F ) H1299 cells were transfected for 24 h as in C followed by TGF- β treatment or left untreated for an additional 24 h. Microscopy images show GFP /Nox4-DN cells along with phospho-FAK staining.

    Journal: British Journal of Cancer

    Article Title: Wild-type and mutant p53 differentially regulate NADPH oxidase 4 in TGF-β-mediated migration of human lung and breast epithelial cells

    doi: 10.1038/bjc.2014.165

    Figure Lengend Snippet: Nox4 and mutant p53 (R280K) modulate TGF- β -induced activation of focal adhesion kinase (pFAK). ( A ) Overexpression of Nox4-DN reduces FAK phosphorylation. MDA-MB-231 cells were transfected with either vector control or V5-tagged Nox4-DN for 24 h followed by TGF- β (5 ng ml −1 ) stimulation for an additional 24 h. Total cell lysates collected for immunoblot analysis using antibodies against Nox4, p53, phospho-FAK (pFAK) and total FAK, and V5. ( B ) MDA-MB-231 cells were transfected with control siRNAs (25 n M ) or p53-specific siRNAs (25 n M ) for 72 h then simulated with TGF- β (5 ng ml −1 ) for 24 h. Protein expression was analysed by immunoblotting total cell lysates with antibodies against Nox4, p53, phospho-FAK (pFAK), and total FAK. ( C ) MDA-MB-231 cells were transfected with Nox4-DN and GFP for 24 h followed by TGF- β treatment for an additional 24 h. Upper panel: GFP (Nox4-DN) cells vs phospho-FAK staining. Lower panel: cells expressing Nox4-DN (arrows) have reduced phospho-FAK staining compared with surrounding non-transfected cells. ( D ) MCF-10A cells were co-transfected with vector alone or Nox4-DN, and p53-WT or p53-R280K plasmids then treated with TGF- β for 24 h. Immunoblotting analysis indicates phospho-FAK, FAK, V5-tagged Nox4-DN, and p53 protein expression. ( E ) p53-null H1299 cells were co-transfected transfected, treated, and analysed for protein expression and FAK phosphorylation as in D . ( F ) H1299 cells were transfected for 24 h as in C followed by TGF- β treatment or left untreated for an additional 24 h. Microscopy images show GFP /Nox4-DN cells along with phospho-FAK staining.

    Article Snippet: Antibodies and immunoblotting analysis Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.

    Techniques: Mutagenesis, Activation Assay, Over Expression, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Expressing, Staining, Microscopy

    Inhibition of Aurora B induces p53 in a breast cancer xenograft model. ( A ) MCF7-Her18 cells were treated with increasing concentrations of AZD1152-HQPA as labeled for 48 h. Antigens for the immunoblots shown are labeled on the left. Mdm2 is indicated as full length (90 kDa) and cleavage product (60 kDa). ( B ) MCF7-Her18 xenografts from nude mice (indicated by mouse number) treated with AZD1152 were immunoblotted for p53 and p53 target genes. The protein expression of Bax, Puma, and Actin was quantified from Western blot films using Image J program. Bax/Actin and Puma/Actin ratios were calculated. Bar graphs and one-way ANOVA statistic analyses with Turkey test were done with GraphPad Prism 5.0c. ( C ) Representative photomicrographs are shown for immunohistochemical staining of p53 in MCF7-Her18 xenograft tumors treated with AZD1152. ( D ) Percentage p53-positive ( Upper ) and average p53 immunostaining intensity ( Lower ) from automated quantitative image analysis of immunohistochemical staining of MCF7-Her18 nude mouse xenografts treated with AZD1152. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Inhibition of Aurora B induces p53 in a breast cancer xenograft model. ( A ) MCF7-Her18 cells were treated with increasing concentrations of AZD1152-HQPA as labeled for 48 h. Antigens for the immunoblots shown are labeled on the left. Mdm2 is indicated as full length (90 kDa) and cleavage product (60 kDa). ( B ) MCF7-Her18 xenografts from nude mice (indicated by mouse number) treated with AZD1152 were immunoblotted for p53 and p53 target genes. The protein expression of Bax, Puma, and Actin was quantified from Western blot films using Image J program. Bax/Actin and Puma/Actin ratios were calculated. Bar graphs and one-way ANOVA statistic analyses with Turkey test were done with GraphPad Prism 5.0c. ( C ) Representative photomicrographs are shown for immunohistochemical staining of p53 in MCF7-Her18 xenograft tumors treated with AZD1152. ( D ) Percentage p53-positive ( Upper ) and average p53 immunostaining intensity ( Lower ) from automated quantitative image analysis of immunohistochemical staining of MCF7-Her18 nude mouse xenografts treated with AZD1152. * P

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Inhibition, Labeling, Western Blot, Mouse Assay, Expressing, Immunohistochemistry, Staining, Immunostaining

    Phosphorylation of p53 by Aurora B inhibits p53 transcriptional activity. ( A ) 293T cells were transfected with fixed amounts of plasmids expressing GFP-p53, p53-Luciferase reporter plasmid (BDS2-3x-luc containing a p53-responsive element), and increasing doses of plasmid expressing GFP-Aurora B. p53 transcriptional activity was measured by luciferase activity. All of the error bars in the bar charts of this figure represent 95% confidence intervals. ( B ) p53-Luciferase reporter assay (BDS2-3x-luc) in 293T cells transfected with increasing dose of shRNA 468-AurB plasmid. ( C ) qRT-PCR analysis was performed to measure p53 and p53 target gene mRNAs in U2OS transfected with vector or Flag-Aurora B. ( D ) Hct116 cells were transfected with Flag-Aurora B, treated with cisplatin (CDDP), and analyzed by FACS after PI staining. Percentage of apoptotic cells (sub-G1 fraction) was plotted. * P . ( E ) H1299 cells were transfected with indicated GFP-p53 phosphorylation site mutants and p53-luciferase reporter plasmid (MDM2-luc containing a p53-responsive element) in the absence (vector) or presence of Flag-Aurora B. Relative luciferase activity of each p53 construct in the presence or absence of Aurora B was measured. Error bars represent 95% confidence intervals. ( F ) qRT-PCR analysis was used to measure p21 mRNA in Hct116 p53 −/− cells transfected with Aurora B and either GFP-p53 or GFP-p53 AAA mutant. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Phosphorylation of p53 by Aurora B inhibits p53 transcriptional activity. ( A ) 293T cells were transfected with fixed amounts of plasmids expressing GFP-p53, p53-Luciferase reporter plasmid (BDS2-3x-luc containing a p53-responsive element), and increasing doses of plasmid expressing GFP-Aurora B. p53 transcriptional activity was measured by luciferase activity. All of the error bars in the bar charts of this figure represent 95% confidence intervals. ( B ) p53-Luciferase reporter assay (BDS2-3x-luc) in 293T cells transfected with increasing dose of shRNA 468-AurB plasmid. ( C ) qRT-PCR analysis was performed to measure p53 and p53 target gene mRNAs in U2OS transfected with vector or Flag-Aurora B. ( D ) Hct116 cells were transfected with Flag-Aurora B, treated with cisplatin (CDDP), and analyzed by FACS after PI staining. Percentage of apoptotic cells (sub-G1 fraction) was plotted. * P . ( E ) H1299 cells were transfected with indicated GFP-p53 phosphorylation site mutants and p53-luciferase reporter plasmid (MDM2-luc containing a p53-responsive element) in the absence (vector) or presence of Flag-Aurora B. Relative luciferase activity of each p53 construct in the presence or absence of Aurora B was measured. Error bars represent 95% confidence intervals. ( F ) qRT-PCR analysis was used to measure p21 mRNA in Hct116 p53 −/− cells transfected with Aurora B and either GFP-p53 or GFP-p53 AAA mutant. * P

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Activity Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Reporter Assay, shRNA, Quantitative RT-PCR, FACS, Staining, Construct, Mutagenesis

    Functional consequences of the interaction between p53 and Aurora B. In interphase, Aurora B phosphorylates p53, leading to its degradation by MDM2-mediated ubiquitination, which down-regulates the expression of cell cycle/apoptosis regulators such as p21 and PUMA. In metaphase, Aurora B associates with p53 at the centromere region to regulate the spindle checkpoint.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Functional consequences of the interaction between p53 and Aurora B. In interphase, Aurora B phosphorylates p53, leading to its degradation by MDM2-mediated ubiquitination, which down-regulates the expression of cell cycle/apoptosis regulators such as p21 and PUMA. In metaphase, Aurora B associates with p53 at the centromere region to regulate the spindle checkpoint.

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Functional Assay, Expressing

    Aurora B colocalizes and directly interacts with p53 in interphase and mitosis. ( A ) MCF7-Her18 cells were stained with DAPI, mouse anti-p53, and rabbit anti-Aurora B antibodies. Confocal immunofluorescence images of cells in anaphase and telophase are shown. ( B ) Schematic diagram to explain the use of Venus fusion proteins for BiFC. ( C ) Plasmids containing the N terminus of Venus fluorescent protein fused to p53 (Venus N-term-p53) and the C terminus of Venus fused to Aurora B (Venus C-term-AurB) were cotransfected in MCF7. Cells were synchronized to prophase by thymidine–nocodazole block. Venus fluorescence is pseudocolored green. ( D ) Deconvolved fluorescent micrograph of a synchronized MCF7 nucleus from C is shown at a high magnification. Arrows indicate p53 and Aurora B direct intermolecular interaction (BiFC) at the centromeres. ( E ) 293T cells transfected with Venus C-term-AurB and Venus N-Term-p53 and immunostained for the CPC member Survivin (red). The merged image shows that BiFC (pseudocolored green) colocalizes with at least some of the Survivin immunofluorescence (red). ( F ) MCF7 cells were transfected with Venus C-term-AurB and Venus N-term-p53 and then treated with thymidine–nocodazole for synchronization. Deconvolved fluorescent micrograph of a synchronized MCF7 nucleus showing colocalization of Venus (Aurora B–p53 interaction) and the centromere marker CENP-A (red).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Aurora B colocalizes and directly interacts with p53 in interphase and mitosis. ( A ) MCF7-Her18 cells were stained with DAPI, mouse anti-p53, and rabbit anti-Aurora B antibodies. Confocal immunofluorescence images of cells in anaphase and telophase are shown. ( B ) Schematic diagram to explain the use of Venus fusion proteins for BiFC. ( C ) Plasmids containing the N terminus of Venus fluorescent protein fused to p53 (Venus N-term-p53) and the C terminus of Venus fused to Aurora B (Venus C-term-AurB) were cotransfected in MCF7. Cells were synchronized to prophase by thymidine–nocodazole block. Venus fluorescence is pseudocolored green. ( D ) Deconvolved fluorescent micrograph of a synchronized MCF7 nucleus from C is shown at a high magnification. Arrows indicate p53 and Aurora B direct intermolecular interaction (BiFC) at the centromeres. ( E ) 293T cells transfected with Venus C-term-AurB and Venus N-Term-p53 and immunostained for the CPC member Survivin (red). The merged image shows that BiFC (pseudocolored green) colocalizes with at least some of the Survivin immunofluorescence (red). ( F ) MCF7 cells were transfected with Venus C-term-AurB and Venus N-term-p53 and then treated with thymidine–nocodazole for synchronization. Deconvolved fluorescent micrograph of a synchronized MCF7 nucleus showing colocalization of Venus (Aurora B–p53 interaction) and the centromere marker CENP-A (red).

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Staining, Immunofluorescence, Bimolecular Fluorescence Complementation Assay, Blocking Assay, Fluorescence, Transfection, Marker

    Aurora B associates with p53 at various phases of the cell cycle. ( A ) Lysates of U2OS cells were IP with either anti-Aurora B (AB2254; Abcam) or anti-p53 antibodies (AB-2) or preimmune IgG (negative control) followed by immunoblotting (IB) with antibodies as labeled to show co-IP of Aurora B and p53. ( B ) GST pull-down assay was performed with combinations of in vitro translated Aurora B, GST, and GST-tagged p53 as labeled. Aurora B that was bound to GST-p53 was detected by IB. Coomassie staining of GST and GST-p53 inputs are in C . In vitro translated Aurora B was detected by immunoblot ( Lower ). ( C ) As presented in B , results of in vitro GST pull-down assay of immunopurified Flag-Aurora B with GST or GST-tagged p53 deletion constructs are shown. The asterisks indicate the stained bands of GST, GST-p53, and GST-p53 deletion mutants. ( D ) Hct116 cells were synchronized to S phase by double thymidine block. Cell samples at labeled time points after release of thymidine block were stained with PI and analyzed by FACS for DNA content. DNA content histograms are shown for the time points as labeled. ( E ) Lysates of synchronized Hct116 cells from D were analyzed by immunoblot with indicated antibodies. Aurora B–p53 interaction at various phases of the cell cycle (as labeled above) was detected by IP with anti-Aurora B antibody followed by IB for p53 and Aurora B (IP:AurB and IB:AurB).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Aurora B associates with p53 at various phases of the cell cycle. ( A ) Lysates of U2OS cells were IP with either anti-Aurora B (AB2254; Abcam) or anti-p53 antibodies (AB-2) or preimmune IgG (negative control) followed by immunoblotting (IB) with antibodies as labeled to show co-IP of Aurora B and p53. ( B ) GST pull-down assay was performed with combinations of in vitro translated Aurora B, GST, and GST-tagged p53 as labeled. Aurora B that was bound to GST-p53 was detected by IB. Coomassie staining of GST and GST-p53 inputs are in C . In vitro translated Aurora B was detected by immunoblot ( Lower ). ( C ) As presented in B , results of in vitro GST pull-down assay of immunopurified Flag-Aurora B with GST or GST-tagged p53 deletion constructs are shown. The asterisks indicate the stained bands of GST, GST-p53, and GST-p53 deletion mutants. ( D ) Hct116 cells were synchronized to S phase by double thymidine block. Cell samples at labeled time points after release of thymidine block were stained with PI and analyzed by FACS for DNA content. DNA content histograms are shown for the time points as labeled. ( E ) Lysates of synchronized Hct116 cells from D were analyzed by immunoblot with indicated antibodies. Aurora B–p53 interaction at various phases of the cell cycle (as labeled above) was detected by IP with anti-Aurora B antibody followed by IB for p53 and Aurora B (IP:AurB and IB:AurB).

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Negative Control, Labeling, Co-Immunoprecipitation Assay, Pull Down Assay, In Vitro, Staining, Construct, Blocking Assay, FACS

    Aurora B phosphorylates p53 at multiple sites resulting in polyubiquitination and degradation by the proteasome. ( A ) H1299 cells were transfected with a fixed amount of plasmid expressing GFP-p53 and an increasing amount of plasmid expressing Flag-Aurora B. Immunoblots for GFP-p53, Flag-Aurora B, and Actin are shown. ( B ) U2OS cells were transfected with Flag-Aurora B or control vector and then exposed to cycloheximide followed by immunoblot for p53 turnover. ( C ) 293T cells were infected with lentivirus expressing shRNA 468 AurB or control lentivirus (shRNA luc). Infected cells were exposed to cycloheximide and then analyzed by immunoblot for p53 turnover. The asterisk (*) represents nonspecific band. ( D ) H1299 cells were cotransfected with GFP-p53 and Flag-Aurora B in the presence and absence of the proteasome inhibitor MG341. ( E ) 293T cells were transfected as indicated and immunoprecipitated with anti-HA antibody followed by immunoblot with anti-GFP antibody to detect ubiquitinated p53. ( F ) 293T cells were transfected as indicated, treated for 24 h before harvest with AZD1152-HQPA, and immunoprecipitated with anti-HA antibody followed by immunoblot with anti-p53 antibody to detect polyubiquitinated p53. ( G ) H1299 cells were transfected with plasmids as indicated and treated with cycloheximide to evaluate the turnover of GFP-tagged p53 or GFP-p53 AAA. Relative remaining GFP-p53 expression value is indicated in a line graph.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Aurora B phosphorylates p53 at multiple sites resulting in polyubiquitination and degradation by the proteasome. ( A ) H1299 cells were transfected with a fixed amount of plasmid expressing GFP-p53 and an increasing amount of plasmid expressing Flag-Aurora B. Immunoblots for GFP-p53, Flag-Aurora B, and Actin are shown. ( B ) U2OS cells were transfected with Flag-Aurora B or control vector and then exposed to cycloheximide followed by immunoblot for p53 turnover. ( C ) 293T cells were infected with lentivirus expressing shRNA 468 AurB or control lentivirus (shRNA luc). Infected cells were exposed to cycloheximide and then analyzed by immunoblot for p53 turnover. The asterisk (*) represents nonspecific band. ( D ) H1299 cells were cotransfected with GFP-p53 and Flag-Aurora B in the presence and absence of the proteasome inhibitor MG341. ( E ) 293T cells were transfected as indicated and immunoprecipitated with anti-HA antibody followed by immunoblot with anti-GFP antibody to detect ubiquitinated p53. ( F ) 293T cells were transfected as indicated, treated for 24 h before harvest with AZD1152-HQPA, and immunoprecipitated with anti-HA antibody followed by immunoblot with anti-p53 antibody to detect polyubiquitinated p53. ( G ) H1299 cells were transfected with plasmids as indicated and treated with cycloheximide to evaluate the turnover of GFP-tagged p53 or GFP-p53 AAA. Relative remaining GFP-p53 expression value is indicated in a line graph.

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Infection, shRNA, Immunoprecipitation

    Aurora B phosphorylates p53 at multiple serine/threonine residues in the DNA binding domain. ( A ) Flag-Aurora B was immunoprecipitated with anti-Flag antibody from lysates of 293T cells transfected with Flag-Aurora B or vector (negative control). The anti-Aurora B immunoblot is shown in A Middle . Coomassie blue-stained SDS/PAGE gel of GST and GST-p53 is shown ( A Bottom ). Phosphorylation resulting from Flag-Aurora B catalyzed in vitro kinase reactions using GST or GST-tagged p53 as the substrate was detected by autoradiography ( A Top ). ( B ) Flag-Aurora B or Flag-Aurora B K106R was expressed in 293T cells and immunoprecipitated to catalyze in vitro kinase reactions as in A . Phosphorylation results are shown in a similar manner to A . ( C ) Recombinant GST-tagged Aurora B was used in an in vitro kinase assay with GST or GST-p53 as the substrate in the presence of an increasing dose of the specific Aurora B inhibitor AZD1152-HQPA. Phosphorylation results are shown in a similar manner to A . ( D ) Recombinant GST-Aurora B was used in an in vitro kinase assay as before with GST-tagged deletion mutants of p53. Phosphorylation results are shown in a similar manner to A . *Phosphorylated GST-p53 (amino acids 160–393) fragment. ( E ) Consensus phosphorylation sequence and Netphos 2.0 scores for potential Aurora B phosphorylation sites in human p53. ( F ) In vitro kinase assay with recombinant GST-Aurora B and various GST-p53 DNA binding domain mutant substrates. Ratio of phosphorylation relative to control is indicated above each lane.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Aurora B kinase phosphorylates and instigates degradation of p53

    doi: 10.1073/pnas.1110287109

    Figure Lengend Snippet: Aurora B phosphorylates p53 at multiple serine/threonine residues in the DNA binding domain. ( A ) Flag-Aurora B was immunoprecipitated with anti-Flag antibody from lysates of 293T cells transfected with Flag-Aurora B or vector (negative control). The anti-Aurora B immunoblot is shown in A Middle . Coomassie blue-stained SDS/PAGE gel of GST and GST-p53 is shown ( A Bottom ). Phosphorylation resulting from Flag-Aurora B catalyzed in vitro kinase reactions using GST or GST-tagged p53 as the substrate was detected by autoradiography ( A Top ). ( B ) Flag-Aurora B or Flag-Aurora B K106R was expressed in 293T cells and immunoprecipitated to catalyze in vitro kinase reactions as in A . Phosphorylation results are shown in a similar manner to A . ( C ) Recombinant GST-tagged Aurora B was used in an in vitro kinase assay with GST or GST-p53 as the substrate in the presence of an increasing dose of the specific Aurora B inhibitor AZD1152-HQPA. Phosphorylation results are shown in a similar manner to A . ( D ) Recombinant GST-Aurora B was used in an in vitro kinase assay as before with GST-tagged deletion mutants of p53. Phosphorylation results are shown in a similar manner to A . *Phosphorylated GST-p53 (amino acids 160–393) fragment. ( E ) Consensus phosphorylation sequence and Netphos 2.0 scores for potential Aurora B phosphorylation sites in human p53. ( F ) In vitro kinase assay with recombinant GST-Aurora B and various GST-p53 DNA binding domain mutant substrates. Ratio of phosphorylation relative to control is indicated above each lane.

    Article Snippet: The following antibodies were used in this study: 14-3-3σ (1433S01; RDI), Actin (A2066; Sigma), Annexin V-FITC (556419; BD Biosciences), Aurora B (Ab2254; Abcam), Bad ( ; BD Biosciences), Bax ( ; BD Transduction), Cyclin A (SC751; Santa Cruz), Cyclin B1 (SC245; Santa Cruz), Cyclin D (MS-2110; Neomarkers), Cyclin E (SC-247; Santa Cruz), Flag (A804-200; Sigma), GFP (SC-9996; Santa Cruz), HA (12CA5; Roche), His (SC-803; Santa Cruz), HH3 (p-S10, 05–817; Upstate), MDM2 (S3813; Santa Cruz), Mouse IgG (488; Alexa; ; Molecular Probes), Mouse IgG (568; Alexa; ; Molecular Probes), p53 for IP (AB-1, PAB1801; Oncogene Science), p53 for IF (SC-6243; Santa Cruz), p53 (610183; BD Biosciences), p53 (p-S315, 2528S0; Cell Signaling), P21 (610233; Transduction Labs), PUMA (SC-28226; Santa Cruz), Rabbit IgG (488; Alexa; A1103; Molecular Probes), rabbit IgG (568; Alexa; A11011; Molecular Probes), and Survivin (2808; Cell Signaling).

    Techniques: Binding Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control, Staining, SDS Page, In Vitro, Autoradiography, Recombinant, Kinase Assay, Sequencing, Mutagenesis

    Keratinizing moderately differentiated SCC revealed a moderate p53 immunopositivity.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: Keratinizing moderately differentiated SCC revealed a moderate p53 immunopositivity.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques:

    Well differentiated SCC revealed intense immunoreactivity for p53.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: Well differentiated SCC revealed intense immunoreactivity for p53.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques:

    FISH analysis using Vysis LSI TP53 Spectrum Orange/CEP 17 Spectrum Green Probe. Three specific green signals fo trisomy 17 and three specific red signals for p53 amplification.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: FISH analysis using Vysis LSI TP53 Spectrum Orange/CEP 17 Spectrum Green Probe. Three specific green signals fo trisomy 17 and three specific red signals for p53 amplification.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques: Fluorescence In Situ Hybridization, Amplification

    Shows chromosome 17 abnormalities, P53 gene mutations in different histological types of the studied SCC cases.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: Shows chromosome 17 abnormalities, P53 gene mutations in different histological types of the studied SCC cases.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques:

    Poorly differentiated SCC showed mild p53 immunoreactivity.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: Poorly differentiated SCC showed mild p53 immunoreactivity.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques:

    Non- keratinizing moderately differentiated SCC showed moderate p53 immunoreactivity.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: Non- keratinizing moderately differentiated SCC showed moderate p53 immunoreactivity.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques:

    Shows the mean ± SD of P53 immunohistochemical expression in different histological grades of the studied SCC cases.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: Shows the mean ± SD of P53 immunohistochemical expression in different histological grades of the studied SCC cases.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques: Immunohistochemistry, Expressing

    FISH analysis using LSI P53 Spectrum Orange/CEP 17 Spectrum Green Probe. One signal for each fluorochrome showing monosomy 17 and p53 deletion.

    Journal: Diagnostic Pathology

    Article Title: Cytogenetic significance of chromosome 17 aberrations and P53 gene mutations as prognostic markers in oral squamous cell carcinoma

    doi: 10.1186/s13000-015-0232-1

    Figure Lengend Snippet: FISH analysis using LSI P53 Spectrum Orange/CEP 17 Spectrum Green Probe. One signal for each fluorochrome showing monosomy 17 and p53 deletion.

    Article Snippet: After the heating steps, the jars were allowed to cool for 30 min. Nonspecific binding was blocked by incubation with 5% bovine serum albumin (BSA) in TBS for 30 min at room temperature, the slides were incubated with the primary anti-P53 at a dilution of 1:200 in antibody diluent (Antibody diluent, Dako Cytomation) overnight at room temperature in a moist chamber.

    Techniques: Fluorescence In Situ Hybridization

    Proposed mechanism of the inhibitory process. The novel polysaccharide derived from algae extract upregulates the phosphorylation of JNK, activates the downstream cascades of p53, caspase-9 and caspase-3, and then leads to the inhibition of cancer cell proliferation, induces cell apoptosis and cell cycle arrest. By contrast, the polysaccharide does not affect the cancer cell migration, which is mediated through the p38 MAPK signaling pathway or the downstream MMP-9/MMP-2. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase.

    Journal: International Journal of Oncology

    Article Title: A novel polysaccharide derived from algae extract inhibits cancer progression via JNK, not via the p38 MAPK signaling pathway

    doi: 10.3892/ijo.2018.4297

    Figure Lengend Snippet: Proposed mechanism of the inhibitory process. The novel polysaccharide derived from algae extract upregulates the phosphorylation of JNK, activates the downstream cascades of p53, caspase-9 and caspase-3, and then leads to the inhibition of cancer cell proliferation, induces cell apoptosis and cell cycle arrest. By contrast, the polysaccharide does not affect the cancer cell migration, which is mediated through the p38 MAPK signaling pathway or the downstream MMP-9/MMP-2. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase.

    Article Snippet: The first reaction was performed using rabbit immunoglobulin (Ig)G antibodies against JNK (cat. no. J4500; 1:2,000), phospho-JNK (cat. no. 07-175; 2 μ g/ml), p53 (cat. no. SAB4503015; 1:500), caspase-9 (cat. no. C7729; 1:300), caspase-3 (cat. no. C9598; 1:3,000) and p38 MAPK (cat. no. SAB4500492; 1:500) (all from Sigma-Aldrich; Merck KGaA) in PBS containing 0.03% Tween-20 for 1 h at room temperature.

    Techniques: Derivative Assay, Inhibition, Migration

    Effect of the novel polysaccharide on cell signaling pathways. MCF-7 cells were pretreated with 5 μ M SP600125 (inhibitor of JNK) for 1 h, and then treated with polysaccharide (100 μ g/ml) for 48 h. (A) p-JNK, p53, caspase-9, caspase-3 and p38 MAPK were determined by western blot analysis. (B) Effect of polysaccharide on protein expression by densitometry. (C) Effect of polysaccharide and SP600125 on protein expression by densitometry. Magnification, ×400. (D) Effect of polysaccharide and SP600125 on ROS generation in MCF-7 cells using H2-dichlorofluorescin diacetate. Data are expressed as the mean ± standard deviation (n=3). ** P

    Journal: International Journal of Oncology

    Article Title: A novel polysaccharide derived from algae extract inhibits cancer progression via JNK, not via the p38 MAPK signaling pathway

    doi: 10.3892/ijo.2018.4297

    Figure Lengend Snippet: Effect of the novel polysaccharide on cell signaling pathways. MCF-7 cells were pretreated with 5 μ M SP600125 (inhibitor of JNK) for 1 h, and then treated with polysaccharide (100 μ g/ml) for 48 h. (A) p-JNK, p53, caspase-9, caspase-3 and p38 MAPK were determined by western blot analysis. (B) Effect of polysaccharide on protein expression by densitometry. (C) Effect of polysaccharide and SP600125 on protein expression by densitometry. Magnification, ×400. (D) Effect of polysaccharide and SP600125 on ROS generation in MCF-7 cells using H2-dichlorofluorescin diacetate. Data are expressed as the mean ± standard deviation (n=3). ** P

    Article Snippet: The first reaction was performed using rabbit immunoglobulin (Ig)G antibodies against JNK (cat. no. J4500; 1:2,000), phospho-JNK (cat. no. 07-175; 2 μ g/ml), p53 (cat. no. SAB4503015; 1:500), caspase-9 (cat. no. C7729; 1:300), caspase-3 (cat. no. C9598; 1:3,000) and p38 MAPK (cat. no. SAB4500492; 1:500) (all from Sigma-Aldrich; Merck KGaA) in PBS containing 0.03% Tween-20 for 1 h at room temperature.

    Techniques: Western Blot, Expressing, Standard Deviation