anti-p53 Search Results


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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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    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 6258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p53
    Effects of 15d-PGJ 2 , J11-Cl and J19 on apoptotic pathways in SKOV3 cells. (A) SKOV3 cells were treated with 15d-PGJ 2 , J11-Cl or J19 at the indicated concentrations for 48 h. The early and late stages of apoptosis were detected on the basis of Annexin V and propidium iodide staining using flow cytometry. (B) Quantification of apoptosis results, presented as the mean ± standard error of the mean of three independent experiments. (C) Effects of 15d-PGJ 2 , J11-Cl and J19 on expression of apoptosis-associated proteins in SKOV3 cells. Changes in Bcl-2 and Bax expression were determined using western blotting and quantified from three independent experiments using densitometry. (D) Effects of 15d-PGJ 2 , J11-Cl and J19 on the expression of apoptosis-associated proteins in SKOV3 cells. Changes in PARP, cleaved caspase-3, cleaved caspase-9, <t>p53</t> and acetylated p53 expression levels were determined relative to expression of β-actin. (E) Quantification of western blot analysis from three independent experiments using densitometry. * P
    P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 97/100, based on 8090 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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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    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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    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
    Mirin induces apoptosis through a <t>p53-dependent</t> mechanism in MNA cells. a , b WB analysis of the indicated proteins and phosphoepitopes ( a ) and real-time PCR quantification of the indicated transcripts ( b ), in LAN5 cells, following mirin treatment for the indicated time points. Blots were probed with β-actin as a loading control. Transcripts expression was normalized on GAPDH levels and reported as fold induction compared to untreated controls. Data obtained by three independent experiments are reported as means ± SD. c , d MTS assay ( c ) and trypan blue exclusion test ( d ) performed in p53 mutant (SK-N-BE), p53 null (LAN1), and p53 wild-type (LAN5) MNA neuroblastoma cell lines, after mirin treatment. e LAN1 cells were transiently transfected with a p53-expressing or an empty plasmid. Apoptosis was evaluated by TUNEL assay (upper panel) and by measuring the amount of the cleaved form of PARP1 (c-PARP) via WB (bottom panel), after 15 and 5 h of mirin treatment, respectively. Average data obtained by three independent TUNEL assays are reported as fold induction compared to controls, ±SD. p was calculated by the ANOVA test. ** p
    Phospho P53, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1299 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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    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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    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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    Cell Signaling Technology Inc antibodies against p53
    <t>p53</t> promoted myocardial necrosis by transcriptionally repressing ARC expression. (A) p53 protein levels in cardiomyocytes treated with 500 μM H 2 O 2 for the indicated time were detected by Western blotting. (B) Overexpression of p53 by adenovirus decreased the protein levels of ARC in cardiomyocytes. (C) Knockdown of p53 partially recovered the protein levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (D) Overexpression of p53 decreased the mRNA levels of ARC. (E) Knockdown of p53 partially recovered the mRNA levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (F) Knockdown of p53 inhibited necrosis in cardiomyocytes exposed to 500 μM H 2 O 2 . Necrosis was detected by PI assay. Error bars represent SEM; *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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    Cell Signaling Technology Inc anti phospho p53 ser15
    <t>p53</t> promoted myocardial necrosis by transcriptionally repressing ARC expression. (A) p53 protein levels in cardiomyocytes treated with 500 μM H 2 O 2 for the indicated time were detected by Western blotting. (B) Overexpression of p53 by adenovirus decreased the protein levels of ARC in cardiomyocytes. (C) Knockdown of p53 partially recovered the protein levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (D) Overexpression of p53 decreased the mRNA levels of ARC. (E) Knockdown of p53 partially recovered the mRNA levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (F) Knockdown of p53 inhibited necrosis in cardiomyocytes exposed to 500 μM H 2 O 2 . Necrosis was detected by PI assay. Error bars represent SEM; *P
    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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    94
    Cell Signaling Technology Inc p53 antibodies
    CR8 attenuates activation of the <t>p53-linked</t> pro-apoptotic pathways following etoposide-induced DNA damage at the mRNA level. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Neurons were collected 24 h after treatment. qPCR quantification of expression of a Noxa, p21, Puma, Apaf-1, and Mcl-1; b promotor region of Noxa and p21; c miR-711 and miR-23a in primary cortical neurons at different time points after treatment. Results of qPCR were normalized to a GAPDH expression; b input DNA; and c U6 snRNA. CR8 attenuates relative expression of PUMA, NOXA, and p21 following 50 μm etoposide treatment ( a ). No change in Apaf-1 relative to controls was observed until 24 h ( a ). Etoposide induced increases in occupancy of p53 in the promoter region of Noxa, and p21 was attenuated by etoposide + CR8 ( b ). n = 3/group for all groups. Etoposide and etoposide + CR8 increased expression of miR-711 and decreased miR-23a compared to control neurons ( c ). n = 6/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p
    P53 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 93 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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    Millipore anti p53 antibody
    (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 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology rabbit anti p53
    <t>p53</t> constrains lung tumour progression in the Kras LA2 - MADM model. ( a ) Schematic of MADM-mediated LOH of p53 in Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM-p53 mice. Stochastic recombination results in removal of one of two duplicate copies of mutant Kras exon1 ( Kras G12D ) and an intervening neo cassette permitting expression of mutant Kras expression and tumour initiation 25 . G2-X MADM recombination, resulting in p53 KO/KO (green, GFP+/tdTomato−) and p53 WT/WT (red, GFP−/tdTomato+) cells, is initiated through tamoxifen activation of Cre ERT2 , permitting localization of Cre to the nucleus. This diagram was adapted with permission from the original MADM schematic 21 . ( b ) Two green tumours (black arrows) were observed on whole-mount analysis of lungs from Kras LA2 , Rosa26-Cre ERT2 /Kras WT ; MADM-p53 mice ( n =8), whereas none were observed in Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM mice (not harbouring p53 mutation, n =10). White arrows denote tumors without fluorescence labelling. We did not detect any red or yellow tumours in either cohort of mice by whole-mount analysis. Merged fluorescence images of green and red filters are shown. ( c ) Histologic section of a tumour in b showed green adenocarcinoma cells adjacent to colourless adenoma cells (predominantly to the right of the line). Some green adenocarcinoma cells (arrow) are intercalating in the adenoma area. Blue, DAPI-stained nuclei. Scale bar, 100 μm. ( d ) Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM-p53 adenoma harbouring rare yellow cells. Blue, DAPI-stained nuclei. Scale bar, 100 μm.
    Rabbit Anti P53, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 338 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Novocastra anti p53
    SVZ cells cultured from ENU-treated <t>p53</t> −/− mice show enhanced self-renewal and impaired differentiation properties. A , Clonal self-renewal in mice of the indicated genotype that were exposed to the carcinogen ENU during the prenatal life. The bar graph indicates the average number of spheres generated by the dissociation of a single sphere after the second (black) or third (gray) passage. B , Note the large size of the ENU-treated p53 −/− sphere even after repeated passaging. C–F , Nestin (green in C , E ), GFAP (red in D , F ), and DAPI (blue in C–F ) immunofluorescence indicating the persistence of large aggregates of immature nestin + cells and abnormal GFAP + cells in cultures from ENU-treated p53 −/− mice ( F ). G–J , Nestin (green in G–I ) and GFAP (red in H–J ) immunofluorescence reveals similar aggregates of nestin + cells ( I ) and astrocytes displaying aberrant morphology (red in J ) only in p53 −/− cells expressing constitutively active Ras (Ras*). Scale bar, 75 μm.
    Anti P53, supplied by Novocastra, used in various techniques. Bioz Stars score: 92/100, based on 362 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson anti p53
    NMDA-PC increases MDM2 protein levels and promotes <t>p53</t> nuclear and cytosolic destabilization. Mouse cortical neurons (9–10 DIV) were exposed to a validated in vitro model of NMDA-PC (Table 1 ). Levels of the E3-ubiquitin ligase MDM2 and p53 were detected by western blotting. GADPH protein levels were used as loading control. A representative western blot image is shown out of three. ( a ) NMDA-PC increased MDM2 levels at 4 hours after OGD. ( b ) This effect occurred in both nuclei and cytosol, together with a decrease in levels of p53 and its target PUMA. Lamin B and GAPDH protein levels were used as nuclear and cytosolic loading control, respectively. ( c ) Confocal images showed that NMDA-PC increased MDM2 levels expression (green) after OGD, which preferentially located in the nucleus (DAPI; blue), and prevented OGD-induced activation of caspase-3 (in red). Scale bar: 20 μm. Relative percentages of neurons with active Caspase-3/MDM2-staining are presented in Supplementary Fig. S1 . Relative protein abundances quantification Fig. 4a and Fig. 4b are presented in Supplementary Fig. S2 and that “full-length blots/gels are presented in Supplementary Fig. S3 .
    Anti P53, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 324 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (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

    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

    Validation of chromatin occupancy and mRNA expression of a selected panel of newly identified p53-target genes. ( a ) ChIP using the anti-p53 antibody DO-1, followed by qPCR, was used to detect relative p53 occupancy in the promoters of 18 selected genes in MCF7 cells treated with nutlin3a, RITA and 5-FU for 8 h. CDKN1A serves as a positive control for p53 binding. ( b ) The mRNA levels of 18 selected genes were detected by qPCR in MCF7 cells treated with nutlin3a

    Journal: Cell Death and Differentiation

    Article Title: Insights into p53 transcriptional function via genome-wide chromatin occupancy and gene expression analysis

    doi: 10.1038/cdd.2012.89

    Figure Lengend Snippet: Validation of chromatin occupancy and mRNA expression of a selected panel of newly identified p53-target genes. ( a ) ChIP using the anti-p53 antibody DO-1, followed by qPCR, was used to detect relative p53 occupancy in the promoters of 18 selected genes in MCF7 cells treated with nutlin3a, RITA and 5-FU for 8 h. CDKN1A serves as a positive control for p53 binding. ( b ) The mRNA levels of 18 selected genes were detected by qPCR in MCF7 cells treated with nutlin3a

    Article Snippet: The proteins were detected using the following antibodies: anti-p53 DO-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Aurora A ab13824 (Abcam, Cambridge, UK) and anti-beta-actin (Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Binding Assay

    Effects of 15d-PGJ 2 , J11-Cl and J19 on apoptotic pathways in SKOV3 cells. (A) SKOV3 cells were treated with 15d-PGJ 2 , J11-Cl or J19 at the indicated concentrations for 48 h. The early and late stages of apoptosis were detected on the basis of Annexin V and propidium iodide staining using flow cytometry. (B) Quantification of apoptosis results, presented as the mean ± standard error of the mean of three independent experiments. (C) Effects of 15d-PGJ 2 , J11-Cl and J19 on expression of apoptosis-associated proteins in SKOV3 cells. Changes in Bcl-2 and Bax expression were determined using western blotting and quantified from three independent experiments using densitometry. (D) Effects of 15d-PGJ 2 , J11-Cl and J19 on the expression of apoptosis-associated proteins in SKOV3 cells. Changes in PARP, cleaved caspase-3, cleaved caspase-9, p53 and acetylated p53 expression levels were determined relative to expression of β-actin. (E) Quantification of western blot analysis from three independent experiments using densitometry. * P

    Journal: International Journal of Oncology

    Article Title: Novel SIRT1 inhibitor 15-deoxy-Δ12,14-prostaglandin J2 and its derivatives exhibit anticancer activity through apoptotic or autophagic cell death pathways in SKOV3 cells

    doi: 10.3892/ijo.2018.4561

    Figure Lengend Snippet: Effects of 15d-PGJ 2 , J11-Cl and J19 on apoptotic pathways in SKOV3 cells. (A) SKOV3 cells were treated with 15d-PGJ 2 , J11-Cl or J19 at the indicated concentrations for 48 h. The early and late stages of apoptosis were detected on the basis of Annexin V and propidium iodide staining using flow cytometry. (B) Quantification of apoptosis results, presented as the mean ± standard error of the mean of three independent experiments. (C) Effects of 15d-PGJ 2 , J11-Cl and J19 on expression of apoptosis-associated proteins in SKOV3 cells. Changes in Bcl-2 and Bax expression were determined using western blotting and quantified from three independent experiments using densitometry. (D) Effects of 15d-PGJ 2 , J11-Cl and J19 on the expression of apoptosis-associated proteins in SKOV3 cells. Changes in PARP, cleaved caspase-3, cleaved caspase-9, p53 and acetylated p53 expression levels were determined relative to expression of β-actin. (E) Quantification of western blot analysis from three independent experiments using densitometry. * P

    Article Snippet: Primary antibodies against SIRT1 (cat. no. 8469; 1:1,000), SIRT2 (cat. no. 12672; 1:1,000), SIRT4 (cat. no. sc-135798; 1:500), SIRT5 (cat. no. 8779; 1:1,000), SIRT6 (cat. no. 8771; 1:1,000), B-cell lymphoma-2 (Bcl-2; cat. no. 15071; 1:500), Bcl-2-associated X protein (Bax; cat. no. 5023; 1:1,000), β-actin (cat. no. 3700; 1:1,000), light chain 3 (LC3; cat. no. 3868; 1:1,000), beclin-1 (cat. no. 4122; 1:1,000), autophagy-related 3 (Atg3; cat. no. 3415; 1:1,000), Atg5 (cat. no. 12994; 1:1,000), Atg7 (cat. no. 8558; 1:1,000), α-tubulin (cat. no. 3873; 1:1,000), cleaved caspase-3 (cat. no. 9661; 1:500), cleaved caspase-9 (cat. no. 7237; 1:1,000), poly(ADP-ribose) polymerase (PARP; cat. no. 9541; 1:1,000) and acetylated p53 (cat. no. 2570; 1:500) were purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Staining, Flow Cytometry, Cytometry, Expressing, Western Blot

    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

    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

    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

    Mirin induces apoptosis through a p53-dependent mechanism in MNA cells. a , b WB analysis of the indicated proteins and phosphoepitopes ( a ) and real-time PCR quantification of the indicated transcripts ( b ), in LAN5 cells, following mirin treatment for the indicated time points. Blots were probed with β-actin as a loading control. Transcripts expression was normalized on GAPDH levels and reported as fold induction compared to untreated controls. Data obtained by three independent experiments are reported as means ± SD. c , d MTS assay ( c ) and trypan blue exclusion test ( d ) performed in p53 mutant (SK-N-BE), p53 null (LAN1), and p53 wild-type (LAN5) MNA neuroblastoma cell lines, after mirin treatment. e LAN1 cells were transiently transfected with a p53-expressing or an empty plasmid. Apoptosis was evaluated by TUNEL assay (upper panel) and by measuring the amount of the cleaved form of PARP1 (c-PARP) via WB (bottom panel), after 15 and 5 h of mirin treatment, respectively. Average data obtained by three independent TUNEL assays are reported as fold induction compared to controls, ±SD. p was calculated by the ANOVA test. ** p

    Journal: Cell Death & Disease

    Article Title: MRE11 inhibition highlights a replication stress-dependent vulnerability of MYCN-driven tumors

    doi: 10.1038/s41419-018-0924-z

    Figure Lengend Snippet: Mirin induces apoptosis through a p53-dependent mechanism in MNA cells. a , b WB analysis of the indicated proteins and phosphoepitopes ( a ) and real-time PCR quantification of the indicated transcripts ( b ), in LAN5 cells, following mirin treatment for the indicated time points. Blots were probed with β-actin as a loading control. Transcripts expression was normalized on GAPDH levels and reported as fold induction compared to untreated controls. Data obtained by three independent experiments are reported as means ± SD. c , d MTS assay ( c ) and trypan blue exclusion test ( d ) performed in p53 mutant (SK-N-BE), p53 null (LAN1), and p53 wild-type (LAN5) MNA neuroblastoma cell lines, after mirin treatment. e LAN1 cells were transiently transfected with a p53-expressing or an empty plasmid. Apoptosis was evaluated by TUNEL assay (upper panel) and by measuring the amount of the cleaved form of PARP1 (c-PARP) via WB (bottom panel), after 15 and 5 h of mirin treatment, respectively. Average data obtained by three independent TUNEL assays are reported as fold induction compared to controls, ±SD. p was calculated by the ANOVA test. ** p

    Article Snippet: Formalin-fixed and paraffin-embedded tissue sections (4 μm thickness) were probed with phospho-histone H2AX (Ser 139) (20E3; Cell Signaling Technology), phospho-p53 (Ser 15) (#9284; Cell Signaling Technology) specific antibodies, according to the manufacturer's instruction of mouse2mouse HRP ready to use kit (MTM001, ScyTek Laboratories, Logan, UT, USA).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, MTS Assay, Mutagenesis, Transfection, Plasmid Preparation, TUNEL Assay

    Schematic representation of the effects of MRE11 inhibition on MYCN-dependent tumors. While promoting an increased proliferation rate, MYCN overexpression also causes high levels of RS, which in turn would be responsible for the accumulation of DNA damage and cell death, if improperly controlled. However, MYCN regulates the expression of MRE11 to keep the deleterious effects of RS in check, thus allowing survival and proliferation of MYCN-driven tumor cells. Under these conditions, MRE11 inhibition leads to accumulation of RS-dependent DNA damage, DDR activation, and p53-dependent cell death

    Journal: Cell Death & Disease

    Article Title: MRE11 inhibition highlights a replication stress-dependent vulnerability of MYCN-driven tumors

    doi: 10.1038/s41419-018-0924-z

    Figure Lengend Snippet: Schematic representation of the effects of MRE11 inhibition on MYCN-dependent tumors. While promoting an increased proliferation rate, MYCN overexpression also causes high levels of RS, which in turn would be responsible for the accumulation of DNA damage and cell death, if improperly controlled. However, MYCN regulates the expression of MRE11 to keep the deleterious effects of RS in check, thus allowing survival and proliferation of MYCN-driven tumor cells. Under these conditions, MRE11 inhibition leads to accumulation of RS-dependent DNA damage, DDR activation, and p53-dependent cell death

    Article Snippet: Formalin-fixed and paraffin-embedded tissue sections (4 μm thickness) were probed with phospho-histone H2AX (Ser 139) (20E3; Cell Signaling Technology), phospho-p53 (Ser 15) (#9284; Cell Signaling Technology) specific antibodies, according to the manufacturer's instruction of mouse2mouse HRP ready to use kit (MTM001, ScyTek Laboratories, Logan, UT, USA).

    Techniques: Inhibition, Over Expression, Expressing, Activation Assay

    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

    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

    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

    p53 promoted myocardial necrosis by transcriptionally repressing ARC expression. (A) p53 protein levels in cardiomyocytes treated with 500 μM H 2 O 2 for the indicated time were detected by Western blotting. (B) Overexpression of p53 by adenovirus decreased the protein levels of ARC in cardiomyocytes. (C) Knockdown of p53 partially recovered the protein levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (D) Overexpression of p53 decreased the mRNA levels of ARC. (E) Knockdown of p53 partially recovered the mRNA levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (F) Knockdown of p53 inhibited necrosis in cardiomyocytes exposed to 500 μM H 2 O 2 . Necrosis was detected by PI assay. Error bars represent SEM; *P

    Journal: Redox Biology

    Article Title: ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening

    doi: 10.1016/j.redox.2018.10.023

    Figure Lengend Snippet: p53 promoted myocardial necrosis by transcriptionally repressing ARC expression. (A) p53 protein levels in cardiomyocytes treated with 500 μM H 2 O 2 for the indicated time were detected by Western blotting. (B) Overexpression of p53 by adenovirus decreased the protein levels of ARC in cardiomyocytes. (C) Knockdown of p53 partially recovered the protein levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (D) Overexpression of p53 decreased the mRNA levels of ARC. (E) Knockdown of p53 partially recovered the mRNA levels of ARC in cardiomyocytes exposed to 500 μM H 2 O 2 . (F) Knockdown of p53 inhibited necrosis in cardiomyocytes exposed to 500 μM H 2 O 2 . Necrosis was detected by PI assay. Error bars represent SEM; *P

    Article Snippet: The anti-p53 antibody (1:1000) was from Cell Signaling Technology (mAb #2524).

    Techniques: Expressing, Western Blot, Over Expression

    ARC protected cardiomyocytes from necrotic cell death in the heart. (A) The protein levels of ARC and p53 in the ischemic heart tissues for the indicated ischemia time were detected by Western blotting. (B) Overexpression of ARC by adenovirus infection attenuated myocardial necrosis during cardiac ischemia/reperfusion (I/R). Mice were subjected to 30 min of LAD ligation followed by 3 h of reperfusion (n = 5/group). Propidium iodide (PI) was injected into the mice to label necrotic cells after the I/R. Representative images of ventricular myocardium sections are shown. Red, PI-positive cardiomyocyte nuclei; blue, 4′,6-diamidino-2-phenylindole–stained nuclei; green, α-actinin; scale bar, 40 µm. (C) Quantitative analysis of PI-positive cells is shown. Error bars represent SEM; *P

    Journal: Redox Biology

    Article Title: ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening

    doi: 10.1016/j.redox.2018.10.023

    Figure Lengend Snippet: ARC protected cardiomyocytes from necrotic cell death in the heart. (A) The protein levels of ARC and p53 in the ischemic heart tissues for the indicated ischemia time were detected by Western blotting. (B) Overexpression of ARC by adenovirus infection attenuated myocardial necrosis during cardiac ischemia/reperfusion (I/R). Mice were subjected to 30 min of LAD ligation followed by 3 h of reperfusion (n = 5/group). Propidium iodide (PI) was injected into the mice to label necrotic cells after the I/R. Representative images of ventricular myocardium sections are shown. Red, PI-positive cardiomyocyte nuclei; blue, 4′,6-diamidino-2-phenylindole–stained nuclei; green, α-actinin; scale bar, 40 µm. (C) Quantitative analysis of PI-positive cells is shown. Error bars represent SEM; *P

    Article Snippet: The anti-p53 antibody (1:1000) was from Cell Signaling Technology (mAb #2524).

    Techniques: Western Blot, Over Expression, Infection, Mouse Assay, Ligation, Injection, Staining

    CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage at the mRNA level. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Neurons were collected 24 h after treatment. qPCR quantification of expression of a Noxa, p21, Puma, Apaf-1, and Mcl-1; b promotor region of Noxa and p21; c miR-711 and miR-23a in primary cortical neurons at different time points after treatment. Results of qPCR were normalized to a GAPDH expression; b input DNA; and c U6 snRNA. CR8 attenuates relative expression of PUMA, NOXA, and p21 following 50 μm etoposide treatment ( a ). No change in Apaf-1 relative to controls was observed until 24 h ( a ). Etoposide induced increases in occupancy of p53 in the promoter region of Noxa, and p21 was attenuated by etoposide + CR8 ( b ). n = 3/group for all groups. Etoposide and etoposide + CR8 increased expression of miR-711 and decreased miR-23a compared to control neurons ( c ). n = 6/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p

    Journal: Cell Death & Disease

    Article Title: Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury

    doi: 10.1038/s41419-018-1156-y

    Figure Lengend Snippet: CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage at the mRNA level. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Neurons were collected 24 h after treatment. qPCR quantification of expression of a Noxa, p21, Puma, Apaf-1, and Mcl-1; b promotor region of Noxa and p21; c miR-711 and miR-23a in primary cortical neurons at different time points after treatment. Results of qPCR were normalized to a GAPDH expression; b input DNA; and c U6 snRNA. CR8 attenuates relative expression of PUMA, NOXA, and p21 following 50 μm etoposide treatment ( a ). No change in Apaf-1 relative to controls was observed until 24 h ( a ). Etoposide induced increases in occupancy of p53 in the promoter region of Noxa, and p21 was attenuated by etoposide + CR8 ( b ). n = 3/group for all groups. Etoposide and etoposide + CR8 increased expression of miR-711 and decreased miR-23a compared to control neurons ( c ). n = 6/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p

    Article Snippet: Immunoprecipitation was performed for 90 min with 1.6 µg of p53 antibodies (Cell Signaling Technology #32532).

    Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Expressing

    CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Twenty-four hours later whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against phospho-ATM, γ-H2A.X, p53, phospho-p53, Puma, Noxa, and p21. Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared with control untreated levels. Cell death occurs in all neurons treated with 50 μm etoposide (phospho-ATM and γ-H2A.X) including an increase in phospho-p53. However, 1 μm CR8 attenuates the etoposide-induced increase in downstream targets of p53 (Puma, Noxa, and p21). n = 3/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p

    Journal: Cell Death & Disease

    Article Title: Comparing effects of CDK inhibition and E2F1/2 ablation on neuronal cell death pathways in vitro and after traumatic brain injury

    doi: 10.1038/s41419-018-1156-y

    Figure Lengend Snippet: CR8 attenuates activation of the p53-linked pro-apoptotic pathways following etoposide-induced DNA damage. Neurons were treated with 50 μm of etoposide ± 1 μm CR8. Twenty-four hours later whole-cell lysates were fractioned on SDS-polyacrylamide gel and immunoblotted with antibodies against phospho-ATM, γ-H2A.X, p53, phospho-p53, Puma, Noxa, and p21. Protein levels were quantified by densitometry, normalized to β-actin, and presented as fold change compared with control untreated levels. Cell death occurs in all neurons treated with 50 μm etoposide (phospho-ATM and γ-H2A.X) including an increase in phospho-p53. However, 1 μm CR8 attenuates the etoposide-induced increase in downstream targets of p53 (Puma, Noxa, and p21). n = 3/group for all groups. Data represent mean ± SEM of one-way ANOVA and Tukey post hoc analysis, * p

    Article Snippet: Immunoprecipitation was performed for 90 min with 1.6 µg of p53 antibodies (Cell Signaling Technology #32532).

    Techniques: Activation Assay

    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

    (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: One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C.

    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: One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C.

    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: One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C.

    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: One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C.

    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: One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C.

    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: One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C.

    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: One milligram of crude nuclear protein extract was incubated overnight with 2 μg of anti-p53 antibody (Ab-7, Calbiochem) or 2 μg of control sheep IgG at 4°C.

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

    p53 constrains lung tumour progression in the Kras LA2 - MADM model. ( a ) Schematic of MADM-mediated LOH of p53 in Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM-p53 mice. Stochastic recombination results in removal of one of two duplicate copies of mutant Kras exon1 ( Kras G12D ) and an intervening neo cassette permitting expression of mutant Kras expression and tumour initiation 25 . G2-X MADM recombination, resulting in p53 KO/KO (green, GFP+/tdTomato−) and p53 WT/WT (red, GFP−/tdTomato+) cells, is initiated through tamoxifen activation of Cre ERT2 , permitting localization of Cre to the nucleus. This diagram was adapted with permission from the original MADM schematic 21 . ( b ) Two green tumours (black arrows) were observed on whole-mount analysis of lungs from Kras LA2 , Rosa26-Cre ERT2 /Kras WT ; MADM-p53 mice ( n =8), whereas none were observed in Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM mice (not harbouring p53 mutation, n =10). White arrows denote tumors without fluorescence labelling. We did not detect any red or yellow tumours in either cohort of mice by whole-mount analysis. Merged fluorescence images of green and red filters are shown. ( c ) Histologic section of a tumour in b showed green adenocarcinoma cells adjacent to colourless adenoma cells (predominantly to the right of the line). Some green adenocarcinoma cells (arrow) are intercalating in the adenoma area. Blue, DAPI-stained nuclei. Scale bar, 100 μm. ( d ) Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM-p53 adenoma harbouring rare yellow cells. Blue, DAPI-stained nuclei. Scale bar, 100 μm.

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p53 constrains lung tumour progression in the Kras LA2 - MADM model. ( a ) Schematic of MADM-mediated LOH of p53 in Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM-p53 mice. Stochastic recombination results in removal of one of two duplicate copies of mutant Kras exon1 ( Kras G12D ) and an intervening neo cassette permitting expression of mutant Kras expression and tumour initiation 25 . G2-X MADM recombination, resulting in p53 KO/KO (green, GFP+/tdTomato−) and p53 WT/WT (red, GFP−/tdTomato+) cells, is initiated through tamoxifen activation of Cre ERT2 , permitting localization of Cre to the nucleus. This diagram was adapted with permission from the original MADM schematic 21 . ( b ) Two green tumours (black arrows) were observed on whole-mount analysis of lungs from Kras LA2 , Rosa26-Cre ERT2 /Kras WT ; MADM-p53 mice ( n =8), whereas none were observed in Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM mice (not harbouring p53 mutation, n =10). White arrows denote tumors without fluorescence labelling. We did not detect any red or yellow tumours in either cohort of mice by whole-mount analysis. Merged fluorescence images of green and red filters are shown. ( c ) Histologic section of a tumour in b showed green adenocarcinoma cells adjacent to colourless adenoma cells (predominantly to the right of the line). Some green adenocarcinoma cells (arrow) are intercalating in the adenoma area. Blue, DAPI-stained nuclei. Scale bar, 100 μm. ( d ) Kras LA2 ,Rosa26-Cre ERT2 /Kras WT ; MADM-p53 adenoma harbouring rare yellow cells. Blue, DAPI-stained nuclei. Scale bar, 100 μm.

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Mouse Assay, Mutagenesis, Expressing, Activation Assay, Fluorescence, Staining

    p53 expression in various stages of lung and pancreatic tumour progression. ( a ) IHC for p53 in LSL-Kras G12D /Kras WT ; p53 LSL-R172H/flox low-grade adenomas and mixed-grade adenocarcinomas revealed p53 staining only in high-grade lung tumour cells. ( b ) IHC for p53 in Pdx1-Cre; LSL-Kras G12D /Kras WT ; p53 LSL-R172H/WT adult pancreas revealed increased p53 expression in higher-grade pancreatic lesions. Arrows show low-grade PanINs. ( c ) A subset of acinar-to-ductal metaplasia (ADM) cells expressed p53 (arrows). ( d ) A subset of low-grade PanIN cells expressed p53 (arrowheads). Scale bars, 100 μm (all).

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p53 expression in various stages of lung and pancreatic tumour progression. ( a ) IHC for p53 in LSL-Kras G12D /Kras WT ; p53 LSL-R172H/flox low-grade adenomas and mixed-grade adenocarcinomas revealed p53 staining only in high-grade lung tumour cells. ( b ) IHC for p53 in Pdx1-Cre; LSL-Kras G12D /Kras WT ; p53 LSL-R172H/WT adult pancreas revealed increased p53 expression in higher-grade pancreatic lesions. Arrows show low-grade PanINs. ( c ) A subset of acinar-to-ductal metaplasia (ADM) cells expressed p53 (arrows). ( d ) A subset of low-grade PanIN cells expressed p53 (arrowheads). Scale bars, 100 μm (all).

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Expressing, Immunohistochemistry, Staining

    p19ARF is expressed in early lesions during pancreatic but not lung tumorigenesis. ( a ) IHC for p19ARF in Pdx1-Cre; LSL-Kras G12D /Kras WT ; p53 LSL-R172H/WT tumours showed expression throughout pancreatic tumorigenesis from early acinar-to-ductal metaplasia (ADM) and PanIN lesions to PDAC. ( b ) IHC for p19ARF exhibited expression in LSL-Kras G12D /Kras WT ; p53 flox/flox lung adenocarcinomas but not adenomas. ( c ) IHC for p19ARF in Pdx1-Cre; LSL-Kras G12D /Kras WT (KC) tumours, lacking p53 mutation, displayed expression throughout pancreatic tumorigenesis. These data suggest that p53 mutation in a does not induce p19ARF by feedback upregulation, as has been previously described 28 . Scale bars, 100 μm (all).

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p19ARF is expressed in early lesions during pancreatic but not lung tumorigenesis. ( a ) IHC for p19ARF in Pdx1-Cre; LSL-Kras G12D /Kras WT ; p53 LSL-R172H/WT tumours showed expression throughout pancreatic tumorigenesis from early acinar-to-ductal metaplasia (ADM) and PanIN lesions to PDAC. ( b ) IHC for p19ARF exhibited expression in LSL-Kras G12D /Kras WT ; p53 flox/flox lung adenocarcinomas but not adenomas. ( c ) IHC for p19ARF in Pdx1-Cre; LSL-Kras G12D /Kras WT (KC) tumours, lacking p53 mutation, displayed expression throughout pancreatic tumorigenesis. These data suggest that p53 mutation in a does not induce p19ARF by feedback upregulation, as has been previously described 28 . Scale bars, 100 μm (all).

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Immunohistochemistry, Expressing, Mutagenesis

    p53 constrains lung tumour progression in the LSL-Kras G12D -MADM model. ( a ) Whole-mount images of K-MADM-p53 mice at various time points following lentiviral Cre administration displaying p53 KO/KO (green, GFP+/tdTomato-), p53 WT/WT (red, GFP-/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) cells within lung tumours. p.i., post infection. ( b ) Lung adenocarcinomas consisting mostly of green cells. ( c ) Mixed-grade tumour consisting of adenoma (left) and adenocarcinoma cells (right). The adenocarcinoma component consists of all green cells. Blue, DAPI-stained nuclei. Scale bars, 200 μm (all).

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p53 constrains lung tumour progression in the LSL-Kras G12D -MADM model. ( a ) Whole-mount images of K-MADM-p53 mice at various time points following lentiviral Cre administration displaying p53 KO/KO (green, GFP+/tdTomato-), p53 WT/WT (red, GFP-/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) cells within lung tumours. p.i., post infection. ( b ) Lung adenocarcinomas consisting mostly of green cells. ( c ) Mixed-grade tumour consisting of adenoma (left) and adenocarcinoma cells (right). The adenocarcinoma component consists of all green cells. Blue, DAPI-stained nuclei. Scale bars, 200 μm (all).

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Mouse Assay, Infection, Staining

    p53 LOH promotes pancreatic tumour progression. ( a ) Pancreas from a 5-week-old Pdx1-Cre-MADM-p53 mouse contains p53 KO/KO (green, GFP+/tdTomato−), p53 WT/WT (red, GFP−/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) acinar cells with normal appearance. ( b ) Low-grade pancreatic intraepithelial neoplasm (PanIN) in an 8-week-old Pdx1-Cre-K-MADM-p53 mouse. ( c ) High-grade PanIN in a 6-week-old Pdx1-Cre-K-MADM-p53 mouse. ( d ) PDAC in an 11-week-old Pdx1-Cre-K-MADM-p53 mouse. ( e ) Liver metastasis in a 10-week-old Pdx1-Cre-K-MADM-p53 mouse harbouring primary PDAC tumours in the pancreas. ( f ) Lymph node metastasis in a 10-week-old Pdx1-Cre-K-MADM-p53 mouse harbouring primary PDAC tumours in the pancreas. ( g ) Kaplan–Meier survival analysis of Kras and p53 mutant mouse models of PDAC. Pdx1-Cre-K-MADM-p53 mice ( n =47, median survival 79 days) harbour intermediate median survival between Pdx1-Cre; LSL-Kras G12D /Kras WT ( KC ) homozygous p53 mutant ( KC; p53 flox/flox ( n =9, median survival 43 days) and KC; p53 LSL-R172H/flox ( n =37, median survival 44 days)) and heterozygous p53 mutant ( KC; p53 flox/WT ( n =7, median survival 154 days) and KC; p53 LSL-R172H/WT ( n =13, median survival 154 days)) models. Pdx1-Cre-K-MADM mice (with wild-type p53, n =20) have prolonged survival similar to that observed with KC mice lacking p53 mutation 12 . Control Pdx1-Cre-MADM-p53 (lacking LSL-Kras G12D , n =21) and K-MADM-p53 (lacking Pdx1-Cre , n =16) mice do not initiate pancreatic tumorigenesis. Blue, DAPI-stained nuclei. Scale bars, 100 μm (all). ( a , b ) were reproduced with permission from MIT.

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p53 LOH promotes pancreatic tumour progression. ( a ) Pancreas from a 5-week-old Pdx1-Cre-MADM-p53 mouse contains p53 KO/KO (green, GFP+/tdTomato−), p53 WT/WT (red, GFP−/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) acinar cells with normal appearance. ( b ) Low-grade pancreatic intraepithelial neoplasm (PanIN) in an 8-week-old Pdx1-Cre-K-MADM-p53 mouse. ( c ) High-grade PanIN in a 6-week-old Pdx1-Cre-K-MADM-p53 mouse. ( d ) PDAC in an 11-week-old Pdx1-Cre-K-MADM-p53 mouse. ( e ) Liver metastasis in a 10-week-old Pdx1-Cre-K-MADM-p53 mouse harbouring primary PDAC tumours in the pancreas. ( f ) Lymph node metastasis in a 10-week-old Pdx1-Cre-K-MADM-p53 mouse harbouring primary PDAC tumours in the pancreas. ( g ) Kaplan–Meier survival analysis of Kras and p53 mutant mouse models of PDAC. Pdx1-Cre-K-MADM-p53 mice ( n =47, median survival 79 days) harbour intermediate median survival between Pdx1-Cre; LSL-Kras G12D /Kras WT ( KC ) homozygous p53 mutant ( KC; p53 flox/flox ( n =9, median survival 43 days) and KC; p53 LSL-R172H/flox ( n =37, median survival 44 days)) and heterozygous p53 mutant ( KC; p53 flox/WT ( n =7, median survival 154 days) and KC; p53 LSL-R172H/WT ( n =13, median survival 154 days)) models. Pdx1-Cre-K-MADM mice (with wild-type p53, n =20) have prolonged survival similar to that observed with KC mice lacking p53 mutation 12 . Control Pdx1-Cre-MADM-p53 (lacking LSL-Kras G12D , n =21) and K-MADM-p53 (lacking Pdx1-Cre , n =16) mice do not initiate pancreatic tumorigenesis. Blue, DAPI-stained nuclei. Scale bars, 100 μm (all). ( a , b ) were reproduced with permission from MIT.

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Mutagenesis, Mouse Assay, Staining

    p53 loss does not significantly have an impact on early lung tumorigenesis. ( a ) Lung adenomas from K-MADM-p53 mice 16 weeks p.i. showing varying degrees of p53 KO/KO (green, GFP+/tdTomato−), p53 WT/WT (red, GFP−/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) cell labelling. Representative images of non-dominant, green-dominant and red-dominant tumours are shown. Blue, DAPI-stained nuclei. Scale bars, 200 μm (all). ( b ) Absolute quantification of observed green-dominant and red-dominant lung adenomas in K-MADM-p53 mice (10–16 weeks p.i., n =5 mice total). Expected numbers are based on 1:1 stoichiometric ratio of green and red cell generation, and stochastic growth thereafter. No statistical difference was observed ( P > 0.05, χ 2 -test). A plurality of tumours (51 of 132) did not exhibit colour dominance. ( c ) Absolute quantification of green and red cells across individual tumours derived from K-MADM-p53 mice evaluated at 10 weeks p.i. ( n =9) and 16 weeks p.i. ( n =8). ( d ) Geometric means (±95% confidence intervals) of green-to-red cell ratio in lung tumours (based on data from c , n =9 at 10 weeks p.i. and n =8 at 16 weeks p.i.). Line represents equal green and red cell numbers (ratio=1). The green-to-red cell ratio is mildly but significantly increased in tumours from 16-week-old mice (* denotes 95% confidence interval does not cross unity). ( e ) No statistically significant correlation between green-to-red cell ratio and total single-labelled (green plus red) cells per tumour was observed ( P > 0.05, linear regression).

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p53 loss does not significantly have an impact on early lung tumorigenesis. ( a ) Lung adenomas from K-MADM-p53 mice 16 weeks p.i. showing varying degrees of p53 KO/KO (green, GFP+/tdTomato−), p53 WT/WT (red, GFP−/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) cell labelling. Representative images of non-dominant, green-dominant and red-dominant tumours are shown. Blue, DAPI-stained nuclei. Scale bars, 200 μm (all). ( b ) Absolute quantification of observed green-dominant and red-dominant lung adenomas in K-MADM-p53 mice (10–16 weeks p.i., n =5 mice total). Expected numbers are based on 1:1 stoichiometric ratio of green and red cell generation, and stochastic growth thereafter. No statistical difference was observed ( P > 0.05, χ 2 -test). A plurality of tumours (51 of 132) did not exhibit colour dominance. ( c ) Absolute quantification of green and red cells across individual tumours derived from K-MADM-p53 mice evaluated at 10 weeks p.i. ( n =9) and 16 weeks p.i. ( n =8). ( d ) Geometric means (±95% confidence intervals) of green-to-red cell ratio in lung tumours (based on data from c , n =9 at 10 weeks p.i. and n =8 at 16 weeks p.i.). Line represents equal green and red cell numbers (ratio=1). The green-to-red cell ratio is mildly but significantly increased in tumours from 16-week-old mice (* denotes 95% confidence interval does not cross unity). ( e ) No statistically significant correlation between green-to-red cell ratio and total single-labelled (green plus red) cells per tumour was observed ( P > 0.05, linear regression).

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Mouse Assay, Staining, Derivative Assay

    p53 constrains pancreatic tumour progression in Pdx1-Cre-K-MADM-p53 mice. ( a ) Representative images of high-grade PanIN and PDAC from 6-week-old Pdx1-Cre-K-MADM-p53 mice show uniform green (GFP+/tdTomato−, p53 KO/KO ) labelling in these advanced lesions. Blue, DAPI-stained nuclei. Scale bars, 100 μm. ( b ) Absolute quantification of high-grade PanINs/PDAC in 6-week-old Pdx1-Cre-K-MADM-p53 mice ( n =5 mice total). Expected numbers are based on 1:1 stoichiometric ratio of green and red cell generation, and were not observed ( P

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p53 constrains pancreatic tumour progression in Pdx1-Cre-K-MADM-p53 mice. ( a ) Representative images of high-grade PanIN and PDAC from 6-week-old Pdx1-Cre-K-MADM-p53 mice show uniform green (GFP+/tdTomato−, p53 KO/KO ) labelling in these advanced lesions. Blue, DAPI-stained nuclei. Scale bars, 100 μm. ( b ) Absolute quantification of high-grade PanINs/PDAC in 6-week-old Pdx1-Cre-K-MADM-p53 mice ( n =5 mice total). Expected numbers are based on 1:1 stoichiometric ratio of green and red cell generation, and were not observed ( P

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Mouse Assay, Staining

    Schematic of MADM system. ( a ) Schematic of MADM-mediated LOH of p53 . Efficient Cre-mediated intra-chromosomal recombination deletes the transcriptional/translational STOP cassette inducing oncogenic Kras activation. Less efficient Cre-mediated inter-chromosomal recombination following DNA replication (during G2 phase) leads to reconstitution of GFP and tdTomato on separate chromosomes before cell division. This diagram was adapted with permission from the original MADM schematic 21 . ( b ) X segregation of chromosomes following mitotic recombination (G2-X) results in genetically distinct daughter cells: p53 KO/KO (green, GFP+/tdTomato−) and p53 WT/WT (red, GFP−/tdTomato+) cells. Z-segregation (G2-Z) leads to the generation of yellow (GFP+/tdTomato+) and colourless (GFP−/TdTomato−) p53 KO/WT cells. ( c ) Cre-mediated inter-chromosomal recombination during G0 or G1 phase results in the production of yellow p53 KO/WT from colourless p53 KO/WT cells. The MADM system affords faithful correlation between the expression of a specific genetically encoded fluorescence marker and genotype.

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: Schematic of MADM system. ( a ) Schematic of MADM-mediated LOH of p53 . Efficient Cre-mediated intra-chromosomal recombination deletes the transcriptional/translational STOP cassette inducing oncogenic Kras activation. Less efficient Cre-mediated inter-chromosomal recombination following DNA replication (during G2 phase) leads to reconstitution of GFP and tdTomato on separate chromosomes before cell division. This diagram was adapted with permission from the original MADM schematic 21 . ( b ) X segregation of chromosomes following mitotic recombination (G2-X) results in genetically distinct daughter cells: p53 KO/KO (green, GFP+/tdTomato−) and p53 WT/WT (red, GFP−/tdTomato+) cells. Z-segregation (G2-Z) leads to the generation of yellow (GFP+/tdTomato+) and colourless (GFP−/TdTomato−) p53 KO/WT cells. ( c ) Cre-mediated inter-chromosomal recombination during G0 or G1 phase results in the production of yellow p53 KO/WT from colourless p53 KO/WT cells. The MADM system affords faithful correlation between the expression of a specific genetically encoded fluorescence marker and genotype.

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Activation Assay, Expressing, Fluorescence, Marker

    Extra- and intra-tumoral dispersal of early lung and pancreatic tumours. ( a ) p53 KO/KO (green, GFP+/tdTomato−) high-grade PanIN with extra-tumoral dispersal of CK19-negative cells (arrows) in a 6-week-old Pdx1-Cre-K-MADM-p53 mouse. ( b ) Stitched confocal image of an entire lung lobe from a 10-week-p.i. K-MADM-p53 mouse reveals multiple low-grade tumours, some harbouring fluorescently labelled cells (arrowheads) and others not (arrows). Most tumours showed dispersed labelling of green and red cells (white arrowheads), whereas some showed clusters of cells (yellow arrowhead). Scale bar, 500 μm. ( c ) Lung adenoma from a 16-week-p.i. K-MADM-p53 mouse shows dispersed green, red and yellow cells. ( d ) Low-grade PanIN from a 6-week-old Pdx1-Cre-K-MADM-p53 mouse shows dispersed green, red and yellow cells. ( e ) Three-dimensional (3D) rendering of multi-photon imaging of a lung adenoma from a 16-week-p.i. K-MADM-p53 mouse showing non-contiguous green (arrows) and red (arrowheads) cells. ( f ) 3D rendering of multi-photon imaging of a low-grade PanIN from a 6-week-old Pdx1-Cre-K-MADM-p53 mouse showing dispersed green (arrows) and red (arrowheads) cells. Blue, DAPI-stained nuclei. Scale bars, 100 μm (all, unless otherwise noted).

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: Extra- and intra-tumoral dispersal of early lung and pancreatic tumours. ( a ) p53 KO/KO (green, GFP+/tdTomato−) high-grade PanIN with extra-tumoral dispersal of CK19-negative cells (arrows) in a 6-week-old Pdx1-Cre-K-MADM-p53 mouse. ( b ) Stitched confocal image of an entire lung lobe from a 10-week-p.i. K-MADM-p53 mouse reveals multiple low-grade tumours, some harbouring fluorescently labelled cells (arrowheads) and others not (arrows). Most tumours showed dispersed labelling of green and red cells (white arrowheads), whereas some showed clusters of cells (yellow arrowhead). Scale bar, 500 μm. ( c ) Lung adenoma from a 16-week-p.i. K-MADM-p53 mouse shows dispersed green, red and yellow cells. ( d ) Low-grade PanIN from a 6-week-old Pdx1-Cre-K-MADM-p53 mouse shows dispersed green, red and yellow cells. ( e ) Three-dimensional (3D) rendering of multi-photon imaging of a lung adenoma from a 16-week-p.i. K-MADM-p53 mouse showing non-contiguous green (arrows) and red (arrowheads) cells. ( f ) 3D rendering of multi-photon imaging of a low-grade PanIN from a 6-week-old Pdx1-Cre-K-MADM-p53 mouse showing dispersed green (arrows) and red (arrowheads) cells. Blue, DAPI-stained nuclei. Scale bars, 100 μm (all, unless otherwise noted).

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Imaging, Staining

    p53 loss promotes the initiation and expansion of low-grade PanINs. ( a ) Low-grade uniform colour PanINs from 6-week-old Pdx1-Cre-K-MADM-p53 mice showing p53 KO/KO (green, GFP+/tdTomato−), p53 WT/WT (red, GFP−/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) cells. Representative images of all-green and all-red PanINs are shown. Blue, DAPI-stained nuclei. Scale bars, 100 μm. ( b ) Absolute quantification of observed all-green and all-red low-grade PanINs from 6-week-old Pdx1-Cre-K-MADM-p53 mice ( n =5 mice total). Expected numbers are based on 1:1 stoichiometric ratio of green and red cell generation, and were not observed ( P

    Journal: Nature Communications

    Article Title: Clonal dynamics following p53 loss of heterozygosity in Kras-driven cancers

    doi: 10.1038/ncomms12685

    Figure Lengend Snippet: p53 loss promotes the initiation and expansion of low-grade PanINs. ( a ) Low-grade uniform colour PanINs from 6-week-old Pdx1-Cre-K-MADM-p53 mice showing p53 KO/KO (green, GFP+/tdTomato−), p53 WT/WT (red, GFP−/tdTomato+) and p53 KO/WT (yellow, GFP+/tdTomato+) cells. Representative images of all-green and all-red PanINs are shown. Blue, DAPI-stained nuclei. Scale bars, 100 μm. ( b ) Absolute quantification of observed all-green and all-red low-grade PanINs from 6-week-old Pdx1-Cre-K-MADM-p53 mice ( n =5 mice total). Expected numbers are based on 1:1 stoichiometric ratio of green and red cell generation, and were not observed ( P

    Article Snippet: Briefly, formalin-fixed paraffin-embedded sections were stained with haematoxylin and eosin or immunostained with rabbit anti-p53 (Novacastra NCL-p53-CM5p, 1:400) or rat anti-p19ARF (Santa Cruz Biotech sc-32748, 1:100) using Impress secondary antibody kits on a Thermo Scientific Autostainer 360.

    Techniques: Mouse Assay, Staining

    SVZ cells cultured from ENU-treated p53 −/− mice show enhanced self-renewal and impaired differentiation properties. A , Clonal self-renewal in mice of the indicated genotype that were exposed to the carcinogen ENU during the prenatal life. The bar graph indicates the average number of spheres generated by the dissociation of a single sphere after the second (black) or third (gray) passage. B , Note the large size of the ENU-treated p53 −/− sphere even after repeated passaging. C–F , Nestin (green in C , E ), GFAP (red in D , F ), and DAPI (blue in C–F ) immunofluorescence indicating the persistence of large aggregates of immature nestin + cells and abnormal GFAP + cells in cultures from ENU-treated p53 −/− mice ( F ). G–J , Nestin (green in G–I ) and GFAP (red in H–J ) immunofluorescence reveals similar aggregates of nestin + cells ( I ) and astrocytes displaying aberrant morphology (red in J ) only in p53 −/− cells expressing constitutively active Ras (Ras*). Scale bar, 75 μm.

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: SVZ cells cultured from ENU-treated p53 −/− mice show enhanced self-renewal and impaired differentiation properties. A , Clonal self-renewal in mice of the indicated genotype that were exposed to the carcinogen ENU during the prenatal life. The bar graph indicates the average number of spheres generated by the dissociation of a single sphere after the second (black) or third (gray) passage. B , Note the large size of the ENU-treated p53 −/− sphere even after repeated passaging. C–F , Nestin (green in C , E ), GFAP (red in D , F ), and DAPI (blue in C–F ) immunofluorescence indicating the persistence of large aggregates of immature nestin + cells and abnormal GFAP + cells in cultures from ENU-treated p53 −/− mice ( F ). G–J , Nestin (green in G–I ) and GFAP (red in H–J ) immunofluorescence reveals similar aggregates of nestin + cells ( I ) and astrocytes displaying aberrant morphology (red in J ) only in p53 −/− cells expressing constitutively active Ras (Ras*). Scale bar, 75 μm.

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: Cell Culture, Mouse Assay, Generated, Passaging, Immunofluorescence, Expressing

    Loss of p53 increases the number of adult neural stem cells and neuroblasts. A , RT-PCR (top) for the detection of p53 mRNA in adult mouse SVZ and Western blot (bottom) for the detection of p53 protein in SVZ protein extracts. Note the weak band detected in the SVZ extracts compared with HeLa cells. Actin was used as loading control. B , Detail of the lateral wall of the ventricle in irradiated (irrad) and not irradiated (not irrad) C57BL/6 wild-type mice. Note that p53 immunoreactivity after irradiation was restricted to cells of the SVZ, whereas the ependymal cells lining the lumen of the ventricles were not immunoreactive. Scale bar, 10 μm. C , Semithin sections of the SVZ in p53 +/+ and p53 −/− mice stained with toluidine blue showing the thickening of the cellular layer. Scale bar, 25 μm. D , Bar graph of the average number of SVZ cells identified per unit length in p53 +/+ (black bars; n = 5) and p53 −/− (gray bars; n = 5) mice. * p

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Loss of p53 increases the number of adult neural stem cells and neuroblasts. A , RT-PCR (top) for the detection of p53 mRNA in adult mouse SVZ and Western blot (bottom) for the detection of p53 protein in SVZ protein extracts. Note the weak band detected in the SVZ extracts compared with HeLa cells. Actin was used as loading control. B , Detail of the lateral wall of the ventricle in irradiated (irrad) and not irradiated (not irrad) C57BL/6 wild-type mice. Note that p53 immunoreactivity after irradiation was restricted to cells of the SVZ, whereas the ependymal cells lining the lumen of the ventricles were not immunoreactive. Scale bar, 10 μm. C , Semithin sections of the SVZ in p53 +/+ and p53 −/− mice stained with toluidine blue showing the thickening of the cellular layer. Scale bar, 25 μm. D , Bar graph of the average number of SVZ cells identified per unit length in p53 +/+ (black bars; n = 5) and p53 −/− (gray bars; n = 5) mice. * p

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Irradiation, Mouse Assay, Staining

    Effect of p53 loss-of-function on neurosphere-forming SVZ cells. A , Representative bright-field picture of secondary neurospheres generated from p53 +/+ and p53 −/− mice. Note the larger size of the spheres in the p53 −/− cultures. B , Quantification of self-renewal in p53 +/+ and p53 −/− high- and low-density cultures. * p

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Effect of p53 loss-of-function on neurosphere-forming SVZ cells. A , Representative bright-field picture of secondary neurospheres generated from p53 +/+ and p53 −/− mice. Note the larger size of the spheres in the p53 −/− cultures. B , Quantification of self-renewal in p53 +/+ and p53 −/− high- and low-density cultures. * p

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: Generated, Mouse Assay

    Loss of p53 increases the number of neuroblasts both in vivo and in vitro . A , Immunofluorescence of the anterolateral superior corner of the SVZ stained with antibodies against PSA-NCAM (red) to identify the presence of neuroblastic migratory A cells. Note the increased immunoreactivity in p53 −/− mice. Scale bar, 250 μm. B , C , Immunocytochemistry of p53 +/+ and p53 −/− SVZ cells differentiated for 7 d in vitro and stained with antibodies against the neuronal marker Tuj1 ( B ) and the oligodendrocyte marker O4 ( C ). Note the increased number of TuJ1 + and O4 + cells in p53 −/− compared with the p53 +/+ cultures. Scale bar, 180 μm. D , Quantification of the results illustrated in B . * p

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Loss of p53 increases the number of neuroblasts both in vivo and in vitro . A , Immunofluorescence of the anterolateral superior corner of the SVZ stained with antibodies against PSA-NCAM (red) to identify the presence of neuroblastic migratory A cells. Note the increased immunoreactivity in p53 −/− mice. Scale bar, 250 μm. B , C , Immunocytochemistry of p53 +/+ and p53 −/− SVZ cells differentiated for 7 d in vitro and stained with antibodies against the neuronal marker Tuj1 ( B ) and the oligodendrocyte marker O4 ( C ). Note the increased number of TuJ1 + and O4 + cells in p53 −/− compared with the p53 +/+ cultures. Scale bar, 180 μm. D , Quantification of the results illustrated in B . * p

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: In Vivo, In Vitro, Immunofluorescence, Staining, Mouse Assay, Immunocytochemistry, Marker

    Loss of p53 confers a proliferative advantage to fast-proliferating population and to quiescent type B cells. A , Immunoreactive BrdU + cells detected in the anterior horn of the SVZ of p53 +/+ and p53 −/− littermates after a 1 h pulse labeling. Scale bar, 120 μm. B , Bar graph representing the number of BrdU + cells per unit area in p53 +/+ (black bar) and p53 −/− (gray bar) mice. The asterisks indicate statistical significance, * p

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Loss of p53 confers a proliferative advantage to fast-proliferating population and to quiescent type B cells. A , Immunoreactive BrdU + cells detected in the anterior horn of the SVZ of p53 +/+ and p53 −/− littermates after a 1 h pulse labeling. Scale bar, 120 μm. B , Bar graph representing the number of BrdU + cells per unit area in p53 +/+ (black bar) and p53 −/− (gray bar) mice. The asterisks indicate statistical significance, * p

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: Labeling, Mouse Assay

    Topographic map showing the heterogeneous distribution of cell types in the wall of the lateral ventricles of p53 −/− mice compared with p53 +/+ . Topographic map of the SVZ determined by electron microscopy analysis and reconstruction of sequential sections. Each cell type is represented by a dot (red, A cells; blue, B cells; green, C cells; v, lateral ventricle); the ependymal cells lining the ventricular lumen are not shown. The boxed area corresponds to the adjacent enlarged semithin sections of the anterolateral superior corner and of the inferior border of the SVZ of p53 +/+ and p53 −/− mice stained with toluidine blue (v, lateral ventricle). Although some individual variability was observed in the length of the SVZ among different mice, no statistically significant difference was detected between the two genotypes.

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Topographic map showing the heterogeneous distribution of cell types in the wall of the lateral ventricles of p53 −/− mice compared with p53 +/+ . Topographic map of the SVZ determined by electron microscopy analysis and reconstruction of sequential sections. Each cell type is represented by a dot (red, A cells; blue, B cells; green, C cells; v, lateral ventricle); the ependymal cells lining the ventricular lumen are not shown. The boxed area corresponds to the adjacent enlarged semithin sections of the anterolateral superior corner and of the inferior border of the SVZ of p53 +/+ and p53 −/− mice stained with toluidine blue (v, lateral ventricle). Although some individual variability was observed in the length of the SVZ among different mice, no statistically significant difference was detected between the two genotypes.

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: Mouse Assay, Electron Microscopy, Staining

    Glioblastoma-like tumors are detected in periventricular locations in adult p53 −/− mice that have been exposed to the mutagen ENU. A , Micrograph of a sagittal section stained with hematoxylin–eosin showing a large tumoral mass (T) in periventricular location. Scale bar, 500 μm. B , Semithin section stained with toluidine blue showing a tumoral mass invading the corpus callosum and displacing the myelinated axons (circle). Scale bar, 75 μm. C , High-magnification view of the tumor, showing anaplastic cells, extensive microvascularization, and hemorrhages typical of glioblastomas. Scale bar, 50 μm. D , Very pleomorphic and anaplastic cells with dark cytoplasm, giant and invaginated nuclei with numerous chromatin clusters, and enormous nucleoli. Scale bar, 10 μm. E , Immunohistochemical analysis of the tumoral mass showing the presence of nestin + (green) and GFAP + (red) cells. DAPI + (blue) was used as nuclear counterstain. Scale bar, 200 μm. F , Increased thymidine incorporation (after 1 h injection) is observed in cells within the tumor and more prominently at the periphery. Scale bar, 10 μm. G , Ultrastructural detail of a tumoral cell (left) contacting a nontumoral astrocyte (right). Note the moderately electron-dense cytoplasm of the tumoral cell (left) compared with the normal astrocyte and the lack of intermediate filaments (rectangle). The tumoral cell is also characterized by the presence of vacuoles in the mitochondria (black arrows) compared with normal cells (white arrows), enlarged Golgi apparatus (black asterisk) compared with control (white asterisk), and abundant and dilated endoplasmic reticulum (arrowheads). Scale bar, 200 nm.

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Glioblastoma-like tumors are detected in periventricular locations in adult p53 −/− mice that have been exposed to the mutagen ENU. A , Micrograph of a sagittal section stained with hematoxylin–eosin showing a large tumoral mass (T) in periventricular location. Scale bar, 500 μm. B , Semithin section stained with toluidine blue showing a tumoral mass invading the corpus callosum and displacing the myelinated axons (circle). Scale bar, 75 μm. C , High-magnification view of the tumor, showing anaplastic cells, extensive microvascularization, and hemorrhages typical of glioblastomas. Scale bar, 50 μm. D , Very pleomorphic and anaplastic cells with dark cytoplasm, giant and invaginated nuclei with numerous chromatin clusters, and enormous nucleoli. Scale bar, 10 μm. E , Immunohistochemical analysis of the tumoral mass showing the presence of nestin + (green) and GFAP + (red) cells. DAPI + (blue) was used as nuclear counterstain. Scale bar, 200 μm. F , Increased thymidine incorporation (after 1 h injection) is observed in cells within the tumor and more prominently at the periphery. Scale bar, 10 μm. G , Ultrastructural detail of a tumoral cell (left) contacting a nontumoral astrocyte (right). Note the moderately electron-dense cytoplasm of the tumoral cell (left) compared with the normal astrocyte and the lack of intermediate filaments (rectangle). The tumoral cell is also characterized by the presence of vacuoles in the mitochondria (black arrows) compared with normal cells (white arrows), enlarged Golgi apparatus (black asterisk) compared with control (white asterisk), and abundant and dilated endoplasmic reticulum (arrowheads). Scale bar, 200 nm.

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: Mouse Assay, Staining, Immunohistochemistry, Injection

    Prenatal exposure of p53 −/− mice to ENU induces the formation of periventricular glioblastoma-like tumors and is associated with the recruitment of the quiescent type B cells to the fast-proliferating compartment. A , Bar graph representing the number of [ 3 H]Thy + cells per unit area in each population of SVZ cells in untreated (gray) and ENU-treated (black) p53 −/− mice. These data indicate that, during ENU treatment, the relative proportions of proliferating C and A cells are decreased, whereas the proportion of proliferating B cells is increased. B , Ultrastructural appearance of activated C cells (asterisks) in ENU-treated p53 −/− mice. Note the presence of two C cells with giant nuclei and dispersed chromatin (left) and one cell with condensed chromatin (right, arrow), likely representing a B cell in transition. Ultrastructurally, C cells are characterized by the presence of large nuclei with invaginations and several nucleoli, a cytoplasm with abundant ribosomes, few mitochondria, endoplasmic reticulum cisternae, and a small Golgi apparatus. Scale bar, 2 μm. Although in physiological conditions the C cells are found in association with A cells, in ENU-treated p53 −/− mice the C cells do not give rise to A cells but continue to divide and form these aggregates. C , The pies indicate the relative contribution of each subpopulation of SVZ cells to the total number of proliferating [ 3 H]Thy + within after a 1 h labeling period. Note the greater proportion of B cells in S-phase in the ENU-treated p53 −/− mice. D , Relative shift of population dynamics before transformation. There is an accumulation of cells with intermediate feature between type B and C cells, with an expansion of the B/C compartment (arrow) at the expenses of the more differentiated neuroblasts.

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Prenatal exposure of p53 −/− mice to ENU induces the formation of periventricular glioblastoma-like tumors and is associated with the recruitment of the quiescent type B cells to the fast-proliferating compartment. A , Bar graph representing the number of [ 3 H]Thy + cells per unit area in each population of SVZ cells in untreated (gray) and ENU-treated (black) p53 −/− mice. These data indicate that, during ENU treatment, the relative proportions of proliferating C and A cells are decreased, whereas the proportion of proliferating B cells is increased. B , Ultrastructural appearance of activated C cells (asterisks) in ENU-treated p53 −/− mice. Note the presence of two C cells with giant nuclei and dispersed chromatin (left) and one cell with condensed chromatin (right, arrow), likely representing a B cell in transition. Ultrastructurally, C cells are characterized by the presence of large nuclei with invaginations and several nucleoli, a cytoplasm with abundant ribosomes, few mitochondria, endoplasmic reticulum cisternae, and a small Golgi apparatus. Scale bar, 2 μm. Although in physiological conditions the C cells are found in association with A cells, in ENU-treated p53 −/− mice the C cells do not give rise to A cells but continue to divide and form these aggregates. C , The pies indicate the relative contribution of each subpopulation of SVZ cells to the total number of proliferating [ 3 H]Thy + within after a 1 h labeling period. Note the greater proportion of B cells in S-phase in the ENU-treated p53 −/− mice. D , Relative shift of population dynamics before transformation. There is an accumulation of cells with intermediate feature between type B and C cells, with an expansion of the B/C compartment (arrow) at the expenses of the more differentiated neuroblasts.

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: Mouse Assay, Labeling, Transformation Assay

    Spontaneous apoptosis is increased in the hyperplastic areas of the p53 −/− SVZ. A , TUNEL assay in vivo in the SVZ of p53 −/− mice, showing the presence of several apoptotic cells (green nuclei) within areas of localized hyperplasia (blue indicates DAPI as nuclear counterstain). B , TUNEL assay in vitro in cells dissociated from p53 +/+ and p53 −/− secondary neurospheres. Apoptotic nuclei are identified by arrows (TUNEL + , green; DAPI, blue as nuclear counterstain). Scale bar, 180 μm.

    Journal: The Journal of Neuroscience

    Article Title: Loss of p53 Induces Changes in the Behavior of Subventricular Zone Cells: Implication for the Genesis of Glial Tumors

    doi: 10.1523/JNEUROSCI.3970-05.2006

    Figure Lengend Snippet: Spontaneous apoptosis is increased in the hyperplastic areas of the p53 −/− SVZ. A , TUNEL assay in vivo in the SVZ of p53 −/− mice, showing the presence of several apoptotic cells (green nuclei) within areas of localized hyperplasia (blue indicates DAPI as nuclear counterstain). B , TUNEL assay in vitro in cells dissociated from p53 +/+ and p53 −/− secondary neurospheres. Apoptotic nuclei are identified by arrows (TUNEL + , green; DAPI, blue as nuclear counterstain). Scale bar, 180 μm.

    Article Snippet: For immunocytochemical and immunohistochemical procedures, the following primary antibodies were used: anti-β-tubulin III (1:500, clone Tuj1; Covance, Berkeley, CA), anti-GFAP [mouse, 1:1000 (Sternberg Monoclonals, Lutherville, MD) or rabbit, 1:1000 (Dako, Glostrup, Denmark)], CC1 antibody (mouse, 1:50; Oncogene, San Diego, CA), anti-PSA-NCAM (mouse, 1:400; AbCys, Paris, France), anti-BrdU (mouse, 1:200; Dako), anti- p53 (rabbit, 1:500 CM5; Novocastra, New Castle upon Tyne, UK), anti-nestin (rat-401, 1:1000; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-Cre (rabbit, 1:3000; Covance).

    Techniques: TUNEL Assay, In Vivo, Mouse Assay, In Vitro

    NMDA-PC increases MDM2 protein levels and promotes p53 nuclear and cytosolic destabilization. Mouse cortical neurons (9–10 DIV) were exposed to a validated in vitro model of NMDA-PC (Table 1 ). Levels of the E3-ubiquitin ligase MDM2 and p53 were detected by western blotting. GADPH protein levels were used as loading control. A representative western blot image is shown out of three. ( a ) NMDA-PC increased MDM2 levels at 4 hours after OGD. ( b ) This effect occurred in both nuclei and cytosol, together with a decrease in levels of p53 and its target PUMA. Lamin B and GAPDH protein levels were used as nuclear and cytosolic loading control, respectively. ( c ) Confocal images showed that NMDA-PC increased MDM2 levels expression (green) after OGD, which preferentially located in the nucleus (DAPI; blue), and prevented OGD-induced activation of caspase-3 (in red). Scale bar: 20 μm. Relative percentages of neurons with active Caspase-3/MDM2-staining are presented in Supplementary Fig. S1 . Relative protein abundances quantification Fig. 4a and Fig. 4b are presented in Supplementary Fig. S2 and that “full-length blots/gels are presented in Supplementary Fig. S3 .

    Journal: Scientific Reports

    Article Title: The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

    doi: 10.1038/s41598-018-19921-x

    Figure Lengend Snippet: NMDA-PC increases MDM2 protein levels and promotes p53 nuclear and cytosolic destabilization. Mouse cortical neurons (9–10 DIV) were exposed to a validated in vitro model of NMDA-PC (Table 1 ). Levels of the E3-ubiquitin ligase MDM2 and p53 were detected by western blotting. GADPH protein levels were used as loading control. A representative western blot image is shown out of three. ( a ) NMDA-PC increased MDM2 levels at 4 hours after OGD. ( b ) This effect occurred in both nuclei and cytosol, together with a decrease in levels of p53 and its target PUMA. Lamin B and GAPDH protein levels were used as nuclear and cytosolic loading control, respectively. ( c ) Confocal images showed that NMDA-PC increased MDM2 levels expression (green) after OGD, which preferentially located in the nucleus (DAPI; blue), and prevented OGD-induced activation of caspase-3 (in red). Scale bar: 20 μm. Relative percentages of neurons with active Caspase-3/MDM2-staining are presented in Supplementary Fig. S1 . Relative protein abundances quantification Fig. 4a and Fig. 4b are presented in Supplementary Fig. S2 and that “full-length blots/gels are presented in Supplementary Fig. S3 .

    Article Snippet: Antibodies used were anti-p53 (554157, BD Biosciences), anti-pp53 (Ser15; 9286, Cell Signaling, Danvers Massachusetts, USA and anti-cleaved caspase-3 (Asp175, 9661, Cell Signaling), anti-p21(556431, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti-PUMA (ab54288) (Abcam, Cambridge, UK), anti-lamin B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-GAPDH (Ambion, Cambridge, UK) overnight at 4 °C.

    Techniques: In Vitro, Western Blot, Expressing, Activation Assay, Staining

    NMDA-PC promotes p53 destabilization throught a posttranslational mechanism. Mouse cortical neurons from fetal wild type (wt) or p53-null mice ( knockou t, ko) (9–10 DIV) were exposed to a validated in vitro model of NMDA-PC (Table 1) . ( a ) OGD induced p53 stabilization at 4 hours after OGD. ( b ) OGD induced the accumulation of phosphorylated p53 form (pp53, Ser 15) and its targets p21 and PUMA as revealed by western blotting. GADPH protein levels were used as loading control. A representative western blot is shown out of four. ( c ) RT-qPCR analysis of p53 gene reveals that p53 mRNA remained unaltered after OGD. ( d ) Fluorescence microphotographs after immunostaining for p53 (in red) and Map-2 (in green) confirmed that NMDA-PC prevented the accumulation of p53 induced by OGD in neurons. Scale bar: 10 μm. Relative percentages of neurons with p53-staining/Map-2-staining are presented in Supplementary Fig. S1 . Relative protein abundances quantification Fig. 2a and Fig. 2b are presented in Supplementary Fig. S2 and that “full-length blots/gels are presented in Supplementary Fig. S3 .

    Journal: Scientific Reports

    Article Title: The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

    doi: 10.1038/s41598-018-19921-x

    Figure Lengend Snippet: NMDA-PC promotes p53 destabilization throught a posttranslational mechanism. Mouse cortical neurons from fetal wild type (wt) or p53-null mice ( knockou t, ko) (9–10 DIV) were exposed to a validated in vitro model of NMDA-PC (Table 1) . ( a ) OGD induced p53 stabilization at 4 hours after OGD. ( b ) OGD induced the accumulation of phosphorylated p53 form (pp53, Ser 15) and its targets p21 and PUMA as revealed by western blotting. GADPH protein levels were used as loading control. A representative western blot is shown out of four. ( c ) RT-qPCR analysis of p53 gene reveals that p53 mRNA remained unaltered after OGD. ( d ) Fluorescence microphotographs after immunostaining for p53 (in red) and Map-2 (in green) confirmed that NMDA-PC prevented the accumulation of p53 induced by OGD in neurons. Scale bar: 10 μm. Relative percentages of neurons with p53-staining/Map-2-staining are presented in Supplementary Fig. S1 . Relative protein abundances quantification Fig. 2a and Fig. 2b are presented in Supplementary Fig. S2 and that “full-length blots/gels are presented in Supplementary Fig. S3 .

    Article Snippet: Antibodies used were anti-p53 (554157, BD Biosciences), anti-pp53 (Ser15; 9286, Cell Signaling, Danvers Massachusetts, USA and anti-cleaved caspase-3 (Asp175, 9661, Cell Signaling), anti-p21(556431, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti-PUMA (ab54288) (Abcam, Cambridge, UK), anti-lamin B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-GAPDH (Ambion, Cambridge, UK) overnight at 4 °C.

    Techniques: Mouse Assay, In Vitro, Western Blot, Quantitative RT-PCR, Fluorescence, Immunostaining, Staining

    IPC attenuated ischemia-induced infarct growth and increases MDM2 protein levels and p53 destabilization in vivo . (a) IPC was generated by 10 min of transient occlusion of the middle cerebral artery previous to tMCAO for 60 min in rat. Sham-operated rats underwent the same surgical procedure except for tMCAO. After 24 hours of tMCAO recovery, rats were euthanized for brain TTC-staining and the results show that ( a ) IPC attenuates tMCAO-induced infarct growth by ( b ) total 60%, cortex 46% and striatum 31% in preconditioned animals (IPC + tMCAO), as compared to non-preconditioned animals prior ischemia insult (SHAM + tMCAO). ( c ) Cortical brain extracts were analyzed by western blotting and ( d , e ) the relative abundance of protein levels was quantified after 24 hours of tMCAO recovery. ( c ) IPC increased MDM2 protein levels ( d ) and promoted p53 destabilization ( e ) in the ipsilateral cortex (patterned area) in preconditioned animals (IPC + tMCAO), as compared to non-preconditioned animals previous tMCAO (SHAM + tMCAO). Representative western blot images are shown out of five. GADPH protein levels were used as loading control. Data are means ± S.E.M. (n = 5 independent western blot assays). Statistical analysis of the results was evaluated by one-way analysis of variance, followed by the least significant difference multiple range test. Student’s t-test was used for comparisons between two groups of values. In all cases, p

    Journal: Scientific Reports

    Article Title: The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

    doi: 10.1038/s41598-018-19921-x

    Figure Lengend Snippet: IPC attenuated ischemia-induced infarct growth and increases MDM2 protein levels and p53 destabilization in vivo . (a) IPC was generated by 10 min of transient occlusion of the middle cerebral artery previous to tMCAO for 60 min in rat. Sham-operated rats underwent the same surgical procedure except for tMCAO. After 24 hours of tMCAO recovery, rats were euthanized for brain TTC-staining and the results show that ( a ) IPC attenuates tMCAO-induced infarct growth by ( b ) total 60%, cortex 46% and striatum 31% in preconditioned animals (IPC + tMCAO), as compared to non-preconditioned animals prior ischemia insult (SHAM + tMCAO). ( c ) Cortical brain extracts were analyzed by western blotting and ( d , e ) the relative abundance of protein levels was quantified after 24 hours of tMCAO recovery. ( c ) IPC increased MDM2 protein levels ( d ) and promoted p53 destabilization ( e ) in the ipsilateral cortex (patterned area) in preconditioned animals (IPC + tMCAO), as compared to non-preconditioned animals previous tMCAO (SHAM + tMCAO). Representative western blot images are shown out of five. GADPH protein levels were used as loading control. Data are means ± S.E.M. (n = 5 independent western blot assays). Statistical analysis of the results was evaluated by one-way analysis of variance, followed by the least significant difference multiple range test. Student’s t-test was used for comparisons between two groups of values. In all cases, p

    Article Snippet: Antibodies used were anti-p53 (554157, BD Biosciences), anti-pp53 (Ser15; 9286, Cell Signaling, Danvers Massachusetts, USA and anti-cleaved caspase-3 (Asp175, 9661, Cell Signaling), anti-p21(556431, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti-PUMA (ab54288) (Abcam, Cambridge, UK), anti-lamin B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-GAPDH (Ambion, Cambridge, UK) overnight at 4 °C.

    Techniques: In Vivo, Generated, Staining, Western Blot

    NMDA-PC prevents ischemia-induced neuronal apoptosis pathway mediated by p53. Cortical neurons from p53 wt or p53 ko mouse were exposed to a validated in vitro model of NMDA-PC (Table 1 ) and neuronal extracts were analyzed by western blotting. ( a ) At 4 hours after OGD, p53 stabilization was observed in wt, but not in p53 ko, neurons, ( b ) which was prevented by NMDA-PC. The lack of p53 totally prevented apoptosis caused by OGD at 4 hours after the ischemic insult, as revealed by ( c ) flow cytometry and active caspase-3 analyzed by ( d ) western blot, ( e ) immunofluorescence and ( f ) fluorimetry analysis. ( c ) The percentage of annexin V-APC stained neurons that were 7AAD negative were considered to be apoptotic (AnnexinV+/7AAD−). GADPH protein levels were used as loading control. ( e ) Fluorescence microphotographs of both wt and ko neurons after immunostaining for active-Caspase-3 (red). Scale bar: 20 μm. ( g ) immunostaining for p53 (red) and Map-2 (green). Scale bar: 15 μm. Data are means ± S.E.M. (n = 3 independent neuronal cultures). Statistical analysis of the results was evaluated by one-way analysis of variance, followed by the least significant difference multiple range test. Student’s t-test was used for comparisons between two groups of values. In all cases, p

    Journal: Scientific Reports

    Article Title: The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

    doi: 10.1038/s41598-018-19921-x

    Figure Lengend Snippet: NMDA-PC prevents ischemia-induced neuronal apoptosis pathway mediated by p53. Cortical neurons from p53 wt or p53 ko mouse were exposed to a validated in vitro model of NMDA-PC (Table 1 ) and neuronal extracts were analyzed by western blotting. ( a ) At 4 hours after OGD, p53 stabilization was observed in wt, but not in p53 ko, neurons, ( b ) which was prevented by NMDA-PC. The lack of p53 totally prevented apoptosis caused by OGD at 4 hours after the ischemic insult, as revealed by ( c ) flow cytometry and active caspase-3 analyzed by ( d ) western blot, ( e ) immunofluorescence and ( f ) fluorimetry analysis. ( c ) The percentage of annexin V-APC stained neurons that were 7AAD negative were considered to be apoptotic (AnnexinV+/7AAD−). GADPH protein levels were used as loading control. ( e ) Fluorescence microphotographs of both wt and ko neurons after immunostaining for active-Caspase-3 (red). Scale bar: 20 μm. ( g ) immunostaining for p53 (red) and Map-2 (green). Scale bar: 15 μm. Data are means ± S.E.M. (n = 3 independent neuronal cultures). Statistical analysis of the results was evaluated by one-way analysis of variance, followed by the least significant difference multiple range test. Student’s t-test was used for comparisons between two groups of values. In all cases, p

    Article Snippet: Antibodies used were anti-p53 (554157, BD Biosciences), anti-pp53 (Ser15; 9286, Cell Signaling, Danvers Massachusetts, USA and anti-cleaved caspase-3 (Asp175, 9661, Cell Signaling), anti-p21(556431, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti-PUMA (ab54288) (Abcam, Cambridge, UK), anti-lamin B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-GAPDH (Ambion, Cambridge, UK) overnight at 4 °C.

    Techniques: In Vitro, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence, Staining, Fluorescence, Immunostaining

    NMDA-PC promotes MDM2-p53 interaction, which prevents ischemia-induced p53 stabilization. Mouse cortical neurons (9–10 DIV) were exposed to a validated in vitro model of NMDA-PC (Table 1 ). Neuronal extracts were obtained and immunoprecipitated with anti-p53 ( a ) and anti-MDM2 ( b ) antibodies. Of the whole cellular extracts used for immunoprecipitation, 10% were loaded on SDS-PAGE as an input control. A representative western blot image is shown of three. ( a , b ) Co-immunoprecipitation assays revealed that p53 co-precipitated with MDM2. ( a , b ) NMDA-PC prior OGD, promotes MDM2-p53 interaction. ( c ) Fluorescence microphotographs revealed that NMDA-PC increases MDM2 protein levels, which appears to be the main effect for NMDA-PC promoting MDM2-p53 interaction at 4 hours after OGD. Although OGD promoted MDM2-p53 interaction, this effect was not enough to prevent OGD-induced p53 stabilization.White arrowheads show MDM2-p53 interaction. Scale bar = 20 μm. Relative percentages of neurons with p53-staining/MDM2-staining are presented in Supplementary Fig. S1 . Relative protein abundances quantification Fig. 5a and Fig. 5b are presented in Supplementary Fig. S2 and that “full-length blots/gels are presented in Supplementary Fig. S3 .

    Journal: Scientific Reports

    Article Title: The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

    doi: 10.1038/s41598-018-19921-x

    Figure Lengend Snippet: NMDA-PC promotes MDM2-p53 interaction, which prevents ischemia-induced p53 stabilization. Mouse cortical neurons (9–10 DIV) were exposed to a validated in vitro model of NMDA-PC (Table 1 ). Neuronal extracts were obtained and immunoprecipitated with anti-p53 ( a ) and anti-MDM2 ( b ) antibodies. Of the whole cellular extracts used for immunoprecipitation, 10% were loaded on SDS-PAGE as an input control. A representative western blot image is shown of three. ( a , b ) Co-immunoprecipitation assays revealed that p53 co-precipitated with MDM2. ( a , b ) NMDA-PC prior OGD, promotes MDM2-p53 interaction. ( c ) Fluorescence microphotographs revealed that NMDA-PC increases MDM2 protein levels, which appears to be the main effect for NMDA-PC promoting MDM2-p53 interaction at 4 hours after OGD. Although OGD promoted MDM2-p53 interaction, this effect was not enough to prevent OGD-induced p53 stabilization.White arrowheads show MDM2-p53 interaction. Scale bar = 20 μm. Relative percentages of neurons with p53-staining/MDM2-staining are presented in Supplementary Fig. S1 . Relative protein abundances quantification Fig. 5a and Fig. 5b are presented in Supplementary Fig. S2 and that “full-length blots/gels are presented in Supplementary Fig. S3 .

    Article Snippet: Antibodies used were anti-p53 (554157, BD Biosciences), anti-pp53 (Ser15; 9286, Cell Signaling, Danvers Massachusetts, USA and anti-cleaved caspase-3 (Asp175, 9661, Cell Signaling), anti-p21(556431, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti-PUMA (ab54288) (Abcam, Cambridge, UK), anti-lamin B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-GAPDH (Ambion, Cambridge, UK) overnight at 4 °C.

    Techniques: In Vitro, Immunoprecipitation, SDS Page, Western Blot, Fluorescence, Staining

    Pharmacological disruption of MDM2-p53 interaction abrogates NMDA-PC-caused neuroprotection after ischemia. Neurons (9–10 DIV) were treated with different concentrations of nutlin-3a (0–10 μM), a potent an specific inhibitor of MDM2 activity, at time periods indicated. ( a ) Nutlin-3a induced a dose-time dependent apoptotic effect in neurons, as compared with untreated neurons. The percentage of annexin V-APC stained neurons that were 7AAD negative were considered to be apoptotic (AnnexinV+/7AAD−). ( b ) As revealed by western blot, treatment with 2 μM nutlin-3a for 2 hours under normoxic conditions (indicated as control) increased MDM2, p53, p21 and PUMA expression levels. A representative western blot image is shown out of three. ( c ) Nutlin-3a-induced p53 stabilization did not cause the activation of caspase-3, when compared with neurons treated with the inductor of apoptosis, etoposide (10 μM, 24 hours; in red). ( d ) Disruption of MDM2-p53 interaction induced by nutlin-3a treatment prevented p53 destabilization caused by NMDA-PC. ( e ) Fluorescence microphotographs of neurons after immunostaining for Map-2 (green) revealed that nutlin-3a abrogates NMDA-PC-induced p53 destabilization and promotes neurite degeneration, as judged by quantification of the average primary neurite length and Map-2-staining area, at 4 hours after OGD. Scale bar: 10 μm. ( f ) MDM2-p53 disruption abrogated NMDA-PC-induced neuroprotection at 4 hours after OGD in wt neurons, ( g ) but not in p53 ko neurons. Data are means ± S.E.M. Statistical analysis of the results was evaluated by one-way analysis of variance, followed by the least significant difference multiple range test. Student’s t-test was used for comparisons between two groups of values. In all cases, p

    Journal: Scientific Reports

    Article Title: The MDM2-p53 pathway is involved in preconditioning-induced neuronal tolerance to ischemia

    doi: 10.1038/s41598-018-19921-x

    Figure Lengend Snippet: Pharmacological disruption of MDM2-p53 interaction abrogates NMDA-PC-caused neuroprotection after ischemia. Neurons (9–10 DIV) were treated with different concentrations of nutlin-3a (0–10 μM), a potent an specific inhibitor of MDM2 activity, at time periods indicated. ( a ) Nutlin-3a induced a dose-time dependent apoptotic effect in neurons, as compared with untreated neurons. The percentage of annexin V-APC stained neurons that were 7AAD negative were considered to be apoptotic (AnnexinV+/7AAD−). ( b ) As revealed by western blot, treatment with 2 μM nutlin-3a for 2 hours under normoxic conditions (indicated as control) increased MDM2, p53, p21 and PUMA expression levels. A representative western blot image is shown out of three. ( c ) Nutlin-3a-induced p53 stabilization did not cause the activation of caspase-3, when compared with neurons treated with the inductor of apoptosis, etoposide (10 μM, 24 hours; in red). ( d ) Disruption of MDM2-p53 interaction induced by nutlin-3a treatment prevented p53 destabilization caused by NMDA-PC. ( e ) Fluorescence microphotographs of neurons after immunostaining for Map-2 (green) revealed that nutlin-3a abrogates NMDA-PC-induced p53 destabilization and promotes neurite degeneration, as judged by quantification of the average primary neurite length and Map-2-staining area, at 4 hours after OGD. Scale bar: 10 μm. ( f ) MDM2-p53 disruption abrogated NMDA-PC-induced neuroprotection at 4 hours after OGD in wt neurons, ( g ) but not in p53 ko neurons. Data are means ± S.E.M. Statistical analysis of the results was evaluated by one-way analysis of variance, followed by the least significant difference multiple range test. Student’s t-test was used for comparisons between two groups of values. In all cases, p

    Article Snippet: Antibodies used were anti-p53 (554157, BD Biosciences), anti-pp53 (Ser15; 9286, Cell Signaling, Danvers Massachusetts, USA and anti-cleaved caspase-3 (Asp175, 9661, Cell Signaling), anti-p21(556431, BD Biosciences), anti-MDM2 (2A10, ab-16895), anti-PUMA (ab54288) (Abcam, Cambridge, UK), anti-lamin B (sc-374015, Santa Cruz Biotechnology, Heidelberg, Germany) and anti-GAPDH (Ambion, Cambridge, UK) overnight at 4 °C.

    Techniques: Activity Assay, Staining, Western Blot, Expressing, Activation Assay, Fluorescence, Immunostaining