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Addgene inc gfp tagged p53

Localization and recruitment kinetic of <t>p53</t> at the DNA damage sites in MCF7 cells. a Fluorescence intensities measured in the control condition (DMSO) and 30 min after 1 µM camptothecin (CPT) or 30 min after 3 Gy γ-rays treatments from different channels for each protein specified along the distance (µm) measured and represented with the white line . The graphs represent the mean fluorescence intensity (y-axis), which is expressed as a function of the distance measured inside the nuclei (distance measured in µm on the x-axis). Nucleus delimited by dotted lines , scale bar 7 µm. b On the left , the fluorescence intensity in MCF-7 nuclei measured for 53BP1 and phospho-Ser 15-p53 in control (DMSO), 40 min after CPT, and 40 min after 3 Gy treatment. Fluorescence intensity (y-axis in the graphs) was measured along the distance (µm) (x-axis in the graphs) represented by the white line . Nuclei delimited by dotted lines , scale bar 7 µm. On the right , higher magnification of a 3 Gy-irradiated MCF7 cell nucleus containing co-localizing 53BP1 and phosphor-ser15-p53 spots. The scale bar shows 3 µm. c Recruitment kinetics for p53-GFP and 53BP1-GFP transiently transfected in MCF7 cells. Cells were microirradiated in the regions of interest ( red dotted circle , diameter 2 µm) with a 355 nm UV laser, and representative pictures were obtained by scanning with WLL 488 nm at the specified time points after irradiation. The scale bar indicates 8 µm

Gfp Tagged P53, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/gfp+tagged+p53/pmc12678470-134-6-13?v=Addgene+inc
Average 93 stars, based on 54 article reviews

gfp tagged p53 - by Bioz Stars, 2026-07

93/100 stars

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1) Product Images from "Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance"

Article Title: Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance

Journal: European Biophysics Journal

doi: 10.1007/s00249-025-01774-8

Localization and recruitment kinetic of p53 at the DNA damage sites in MCF7 cells. a Fluorescence intensities measured in the control condition (DMSO) and 30 min after 1 µM camptothecin (CPT) or 30 min after 3 Gy γ-rays treatments from different channels for each protein specified along the distance (µm) measured and represented with the white line . The graphs represent the mean fluorescence intensity (y-axis), which is expressed as a function of the distance measured inside the nuclei (distance measured in µm on the x-axis). Nucleus delimited by dotted lines , scale bar 7 µm. b On the left , the fluorescence intensity in MCF-7 nuclei measured for 53BP1 and phospho-Ser 15-p53 in control (DMSO), 40 min after CPT, and 40 min after 3 Gy treatment. Fluorescence intensity (y-axis in the graphs) was measured along the distance (µm) (x-axis in the graphs) represented by the white line . Nuclei delimited by dotted lines , scale bar 7 µm. On the right , higher magnification of a 3 Gy-irradiated MCF7 cell nucleus containing co-localizing 53BP1 and phosphor-ser15-p53 spots. The scale bar shows 3 µm. c Recruitment kinetics for p53-GFP and 53BP1-GFP transiently transfected in MCF7 cells. Cells were microirradiated in the regions of interest ( red dotted circle , diameter 2 µm) with a 355 nm UV laser, and representative pictures were obtained by scanning with WLL 488 nm at the specified time points after irradiation. The scale bar indicates 8 µm

Figure Legend Snippet: Localization and recruitment kinetic of p53 at the DNA damage sites in MCF7 cells. a Fluorescence intensities measured in the control condition (DMSO) and 30 min after 1 µM camptothecin (CPT) or 30 min after 3 Gy γ-rays treatments from different channels for each protein specified along the distance (µm) measured and represented with the white line . The graphs represent the mean fluorescence intensity (y-axis), which is expressed as a function of the distance measured inside the nuclei (distance measured in µm on the x-axis). Nucleus delimited by dotted lines , scale bar 7 µm. b On the left , the fluorescence intensity in MCF-7 nuclei measured for 53BP1 and phospho-Ser 15-p53 in control (DMSO), 40 min after CPT, and 40 min after 3 Gy treatment. Fluorescence intensity (y-axis in the graphs) was measured along the distance (µm) (x-axis in the graphs) represented by the white line . Nuclei delimited by dotted lines , scale bar 7 µm. On the right , higher magnification of a 3 Gy-irradiated MCF7 cell nucleus containing co-localizing 53BP1 and phosphor-ser15-p53 spots. The scale bar shows 3 µm. c Recruitment kinetics for p53-GFP and 53BP1-GFP transiently transfected in MCF7 cells. Cells were microirradiated in the regions of interest ( red dotted circle , diameter 2 µm) with a 355 nm UV laser, and representative pictures were obtained by scanning with WLL 488 nm at the specified time points after irradiation. The scale bar indicates 8 µm

Techniques Used: Fluorescence, Control, Irradiation, Transfection

γH2AX and 53BP1 detection in 3 Gy-irradiated p53 wild-type and mutant cell lines. a Representative images of γH2AX foci for the cell line considered; cell nuclei are delimited by dotted lines. Scale bars 15 µm. b Bar chart shows the quantification of the number of foci for γH2AX per cell nucleus. c Representative images of 53BP1 foci, each cell nuclei are delimited by dotted lines. The scale bar shows 10 µm. d 53BP1 number of foci measured in each cell nucleus. The mean ± S.D. was calculated from three biological replicates ( N > 500 cells). Statistical significance is shown in Supplementary Table 1

Figure Legend Snippet: γH2AX and 53BP1 detection in 3 Gy-irradiated p53 wild-type and mutant cell lines. a Representative images of γH2AX foci for the cell line considered; cell nuclei are delimited by dotted lines. Scale bars 15 µm. b Bar chart shows the quantification of the number of foci for γH2AX per cell nucleus. c Representative images of 53BP1 foci, each cell nuclei are delimited by dotted lines. The scale bar shows 10 µm. d 53BP1 number of foci measured in each cell nucleus. The mean ± S.D. was calculated from three biological replicates ( N > 500 cells). Statistical significance is shown in Supplementary Table 1

Techniques Used: Irradiation, Mutagenesis

$BRCA1 recruitment at the DNA damage sites, γH2AX foci formation, and survival assay in p53 wild-type and mutant cell lines exposed to a multifractionated dose of radiation (3 × 2 Gy). Panel a shows representative images of BRCA1 foci in non-irradiated cells and cells after 3 Gy irradiation. Each cell nucleus is delimited by dotted lines. Scale bars show 15 µm. b Quantification of BRCA1 foci number. Red bars represent the mean ± S.D. ( N > 150 cells). c Immunofluorescence panel for γH2AX nuclear pattern after cellular exposition to multifractionated radiation ( white arrows denote micronuclei-like structures). d Quantification of the foci number studied for γH2AX ( N > 1000 cells). e Survival assay of p53 wild-type and mutant cell lines after single or multifractionated doses of radiation. f Quantification of the number of colonies in each cell line for each condition. Statistics were applied by one-way ANOVA with multiple comparisons (adjusted with Tukey’s correction), mean ± S.D. of two biological repetitions, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001$
Figure Legend Snippet: BRCA1 recruitment at the DNA damage sites, γH2AX foci formation, and survival assay in p53 wild-type and mutant cell lines exposed to a multifractionated dose of radiation (3 × 2 Gy). Panel a shows representative images of BRCA1 foci in non-irradiated cells and cells after 3 Gy irradiation. Each cell nucleus is delimited by dotted lines. Scale bars show 15 µm. b Quantification of BRCA1 foci number. Red bars represent the mean ± S.D. ( N > 150 cells). c Immunofluorescence panel for γH2AX nuclear pattern after cellular exposition to multifractionated radiation ( white arrows denote micronuclei-like structures). d Quantification of the foci number studied for γH2AX ( N > 1000 cells). e Survival assay of p53 wild-type and mutant cell lines after single or multifractionated doses of radiation. f Quantification of the number of colonies in each cell line for each condition. Statistics were applied by one-way ANOVA with multiple comparisons (adjusted with Tukey’s correction), mean ± S.D. of two biological repetitions, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Techniques Used: Clonogenic Cell Survival Assay, Mutagenesis, Irradiation, Immunofluorescence

Image Search Results

Journal: European Biophysics Journal

Article Title: Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance

doi: 10.1007/s00249-025-01774-8

Figure Lengend Snippet: Localization and recruitment kinetic of p53 at the DNA damage sites in MCF7 cells. a Fluorescence intensities measured in the control condition (DMSO) and 30 min after 1 µM camptothecin (CPT) or 30 min after 3 Gy γ-rays treatments from different channels for each protein specified along the distance (µm) measured and represented with the white line . The graphs represent the mean fluorescence intensity (y-axis), which is expressed as a function of the distance measured inside the nuclei (distance measured in µm on the x-axis). Nucleus delimited by dotted lines , scale bar 7 µm. b On the left , the fluorescence intensity in MCF-7 nuclei measured for 53BP1 and phospho-Ser 15-p53 in control (DMSO), 40 min after CPT, and 40 min after 3 Gy treatment. Fluorescence intensity (y-axis in the graphs) was measured along the distance (µm) (x-axis in the graphs) represented by the white line . Nuclei delimited by dotted lines , scale bar 7 µm. On the right , higher magnification of a 3 Gy-irradiated MCF7 cell nucleus containing co-localizing 53BP1 and phosphor-ser15-p53 spots. The scale bar shows 3 µm. c Recruitment kinetics for p53-GFP and 53BP1-GFP transiently transfected in MCF7 cells. Cells were microirradiated in the regions of interest ( red dotted circle , diameter 2 µm) with a 355 nm UV laser, and representative pictures were obtained by scanning with WLL 488 nm at the specified time points after irradiation. The scale bar indicates 8 µm

Article Snippet: MCF-7 cells were transiently transfected with GFP-tagged p53 (a gift from Tyler Jacks, Addgene 12091) (Boyd et al. ) and GFP-tagged 53BP1 (a gift from Daniel Durocher, Addgene 60813) (Fradet-Turcotte et al. ) plasmids using Metafectene pro Kit (Biotex Laboratories, GimbH, Munchen, Germany) and following the manufacturer’s instructions.

Techniques: Fluorescence, Control, Irradiation, Transfection

Journal: European Biophysics Journal

Article Title: Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance

doi: 10.1007/s00249-025-01774-8

Figure Lengend Snippet: γH2AX and 53BP1 detection in 3 Gy-irradiated p53 wild-type and mutant cell lines. a Representative images of γH2AX foci for the cell line considered; cell nuclei are delimited by dotted lines. Scale bars 15 µm. b Bar chart shows the quantification of the number of foci for γH2AX per cell nucleus. c Representative images of 53BP1 foci, each cell nuclei are delimited by dotted lines. The scale bar shows 10 µm. d 53BP1 number of foci measured in each cell nucleus. The mean ± S.D. was calculated from three biological replicates ( N > 500 cells). Statistical significance is shown in Supplementary Table 1

Techniques: Irradiation, Mutagenesis

Journal: European Biophysics Journal

Article Title: Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance

doi: 10.1007/s00249-025-01774-8

Figure Lengend Snippet: BRCA1 recruitment at the DNA damage sites, γH2AX foci formation, and survival assay in p53 wild-type and mutant cell lines exposed to a multifractionated dose of radiation (3 × 2 Gy). Panel a shows representative images of BRCA1 foci in non-irradiated cells and cells after 3 Gy irradiation. Each cell nucleus is delimited by dotted lines. Scale bars show 15 µm. b Quantification of BRCA1 foci number. Red bars represent the mean ± S.D. ( N > 150 cells). c Immunofluorescence panel for γH2AX nuclear pattern after cellular exposition to multifractionated radiation ( white arrows denote micronuclei-like structures). d Quantification of the foci number studied for γH2AX ( N > 1000 cells). e Survival assay of p53 wild-type and mutant cell lines after single or multifractionated doses of radiation. f Quantification of the number of colonies in each cell line for each condition. Statistics were applied by one-way ANOVA with multiple comparisons (adjusted with Tukey’s correction), mean ± S.D. of two biological repetitions, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Techniques: Clonogenic Cell Survival Assay, Mutagenesis, Irradiation, Immunofluorescence

System x C - expression is inversely correlated with p53 functional status. ( A ) Sanger sequencing was performed on three different PDX GBM lines. Sequencing revealed different p53 statuses whereby GBM12 cells were p53-null, GBM14 cells were p53 wild-type, and GBM22 cells were mutant p53 (R273C). Transcript analysis and protein analysis results are also summarized in this table from experiments completed in the subsequent panels. ( B ) RT-qPCR and Western blot analysis of SLC7A11 and xCT expression in all three xenolines from ( A ). ( C ) RT-qPCR and Western blot analysis of TP53 and p53 expression in all three xenolines from ( A ). ( D ) RT-qPCR and Western blot analysis of P21 and p21 expression in all three xenolines from ( A ). All RT-qPCR and Western blot experiments are represented by at least three biological replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed. Error bars represent the mean ± standard error of the mean (S.E.M.). **** p < 0.0001, *** p < 0.001, ** p < 0.01. ( E , F ) All three xenolines from ( A ) were subjected to ICC analysis for xCT ( E ) and p53 ( F ) (green) and counter-stained with nuclear DAPI (blue). Note the characteristic accumulation of mutant p53 in the nucleus of GMB22 cells (( F ), right panels). Scale bar = 20 µm.

Journal: Cancers

Article Title: Transcriptional Regulation of Amino Acid Transport in Glioblastoma Multiforme

doi: 10.3390/cancers13246169

Figure Lengend Snippet: System x C - expression is inversely correlated with p53 functional status. ( A ) Sanger sequencing was performed on three different PDX GBM lines. Sequencing revealed different p53 statuses whereby GBM12 cells were p53-null, GBM14 cells were p53 wild-type, and GBM22 cells were mutant p53 (R273C). Transcript analysis and protein analysis results are also summarized in this table from experiments completed in the subsequent panels. ( B ) RT-qPCR and Western blot analysis of SLC7A11 and xCT expression in all three xenolines from ( A ). ( C ) RT-qPCR and Western blot analysis of TP53 and p53 expression in all three xenolines from ( A ). ( D ) RT-qPCR and Western blot analysis of P21 and p21 expression in all three xenolines from ( A ). All RT-qPCR and Western blot experiments are represented by at least three biological replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed. Error bars represent the mean ± standard error of the mean (S.E.M.). **** p < 0.0001, *** p < 0.001, ** p < 0.01. ( E , F ) All three xenolines from ( A ) were subjected to ICC analysis for xCT ( E ) and p53 ( F ) (green) and counter-stained with nuclear DAPI (blue). Note the characteristic accumulation of mutant p53 in the nucleus of GMB22 cells (( F ), right panels). Scale bar = 20 µm.

Article Snippet: GBM12 cells were transduced with either a control lentivirus that expressed green fluorescent protein (GFP) (Origene; (Rockville, MD, USA, #PS100093V) or an experimental lentivirus that over-expressed p53 conjugated to GFP (Origene #RC200003L4V).

Techniques: Expressing, Functional Assay, Sequencing, Mutagenesis, Quantitative RT-PCR, Western Blot, Staining

SLC7A11 expression is inversely related to p53 expression in patient database samples. ( A ) Correlation analysis between TP53 and SLC7A11 expression was performed using the GlioVis portal and the TCGA database. ( B ) A gene search for “TP53” and “SLC7A11” demonstrate various features such as the “Leading Edge”, “Infiltrating Tumor”, “Cellular Tumor”, “Perinecrotic Zone”, or “Microvascular proliferation”. These features display various relationships between TP53 and SLC7A11 . Two-way ANOVA, with a multiple comparisons post hoc Šidák’s test was performed. Error bars are graphed with the mean ± S.E.M. **** p < 0.0001, ** p < 0.01.

Journal: Cancers

Article Title: Transcriptional Regulation of Amino Acid Transport in Glioblastoma Multiforme

doi: 10.3390/cancers13246169

Figure Lengend Snippet: SLC7A11 expression is inversely related to p53 expression in patient database samples. ( A ) Correlation analysis between TP53 and SLC7A11 expression was performed using the GlioVis portal and the TCGA database. ( B ) A gene search for “TP53” and “SLC7A11” demonstrate various features such as the “Leading Edge”, “Infiltrating Tumor”, “Cellular Tumor”, “Perinecrotic Zone”, or “Microvascular proliferation”. These features display various relationships between TP53 and SLC7A11 . Two-way ANOVA, with a multiple comparisons post hoc Šidák’s test was performed. Error bars are graphed with the mean ± S.E.M. **** p < 0.0001, ** p < 0.01.

Techniques: Expressing

p53 is a molecular switch for xCT expression and function. ( A ) A graphic summary of primer location for ChIP experiments. Gels show amplification of SLC7A11 and P21 after ChIP in three PDX lines. In wild-type p53 tumor cells (GBM14), there is a clear band for SLC7A11 and P21 over the IgG control, unlike in p53-null (GBM12) and mutant (GBM22) lines. Multiple ChIP experiments were performed; this is a representative gel. ( B , C ) All RT-qPCR experiments represent at least three biological replicates. An unpaired two-tailed t-test was performed, and error bars represent the mean ± S.E.M. ** p < 0.005.

Journal: Cancers

Article Title: Transcriptional Regulation of Amino Acid Transport in Glioblastoma Multiforme

doi: 10.3390/cancers13246169

Figure Lengend Snippet: p53 is a molecular switch for xCT expression and function. ( A ) A graphic summary of primer location for ChIP experiments. Gels show amplification of SLC7A11 and P21 after ChIP in three PDX lines. In wild-type p53 tumor cells (GBM14), there is a clear band for SLC7A11 and P21 over the IgG control, unlike in p53-null (GBM12) and mutant (GBM22) lines. Multiple ChIP experiments were performed; this is a representative gel. ( B , C ) All RT-qPCR experiments represent at least three biological replicates. An unpaired two-tailed t-test was performed, and error bars represent the mean ± S.E.M. ** p < 0.005.

Techniques: Expressing, Amplification, Control, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

p53 over-expression decreases in vitro GBM xCT expression and glutamate release. ( A ) Fluorescent images were acquired with a Nikon Ti2 microscope to document the successful infection of GBM12 cells with either a lentivirus over-expressing GFP (left) or p53 conjugated to GFP (right); 10x magnification. Densely expressed GFP in the P53-GFP over-expressing tumor cells (arrows). ( B , C ) Western blot analysis of transduced tumor cells shows P53 overexpression in GBM12 cells ( B ) reduces high xCT expression ( C ) compared to non-transduced and GFP controls. All experiments represent at least three biological replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed. Error bars represent the mean ± S.E.M. *** p < 0.005, ** p < 0.01 ( D ) Media were taken from transduced cells analyzed in ( B , C ) and the glutamate content was measured. p53 over-expression reduced glutamate release in GBM12 cells. An unpaired, two-tailed t -test was performed. Error bars represent the mean ± S.E.M. Experiments represent at least three biological replicates. * p < 0.05.

Journal: Cancers

Article Title: Transcriptional Regulation of Amino Acid Transport in Glioblastoma Multiforme

doi: 10.3390/cancers13246169

Figure Lengend Snippet: p53 over-expression decreases in vitro GBM xCT expression and glutamate release. ( A ) Fluorescent images were acquired with a Nikon Ti2 microscope to document the successful infection of GBM12 cells with either a lentivirus over-expressing GFP (left) or p53 conjugated to GFP (right); 10x magnification. Densely expressed GFP in the P53-GFP over-expressing tumor cells (arrows). ( B , C ) Western blot analysis of transduced tumor cells shows P53 overexpression in GBM12 cells ( B ) reduces high xCT expression ( C ) compared to non-transduced and GFP controls. All experiments represent at least three biological replicates. An ordinary one-way ANOVA with Tukey’s multiple comparisons test was performed. Error bars represent the mean ± S.E.M. *** p < 0.005, ** p < 0.01 ( D ) Media were taken from transduced cells analyzed in ( B , C ) and the glutamate content was measured. p53 over-expression reduced glutamate release in GBM12 cells. An unpaired, two-tailed t -test was performed. Error bars represent the mean ± S.E.M. Experiments represent at least three biological replicates. * p < 0.05.

Techniques: Over Expression, In Vitro, Expressing, Microscopy, Infection, Western Blot, Two Tailed Test

Mutant p53 chemical restoration mitigates SXC-mediated glioma biology in vivo. ( A ) A schematic representing the tumor implantation and dosing regimen for our PRIMA-1 Met in vivo flank experiment. There were 8–10 animals per dosing group. ( B ) Tumor volumes were measured in animals during routine animal health monitoring. Tumor growth decreased in the PRIMA-1 Met -treated animals compared to saline controls. A two-way ANOVA with Šidák’s multiple comparisons test was performed. Error bars represent S.E.M. **** p < 0.0001, *** p < 0.001, * p < 0.05. ( C ) The rate of tumor growth from ( B ) plotted for both treatment groups also shows that the growth rate decreased in the PRIMA-1 Met -treated animals compared to saline controls. An unpaired t-test was performed. Error bars represent the mean ± S.E.M. ** p < 0.005 ( D , E ) Western blot analysis of harvested tumor tissue was performed after the conclusion of the dosing experiment. Both p53 and xCT protein levels were decreased in PRIMA-1 Met -treated animals compared to saline controls. An unpaired, one-tailed t-test was performed. ** p < 0.01, * p < 0.05.

Journal: Cancers

Article Title: Transcriptional Regulation of Amino Acid Transport in Glioblastoma Multiforme

doi: 10.3390/cancers13246169

Figure Lengend Snippet: Mutant p53 chemical restoration mitigates SXC-mediated glioma biology in vivo. ( A ) A schematic representing the tumor implantation and dosing regimen for our PRIMA-1 Met in vivo flank experiment. There were 8–10 animals per dosing group. ( B ) Tumor volumes were measured in animals during routine animal health monitoring. Tumor growth decreased in the PRIMA-1 Met -treated animals compared to saline controls. A two-way ANOVA with Šidák’s multiple comparisons test was performed. Error bars represent S.E.M. **** p < 0.0001, *** p < 0.001, * p < 0.05. ( C ) The rate of tumor growth from ( B ) plotted for both treatment groups also shows that the growth rate decreased in the PRIMA-1 Met -treated animals compared to saline controls. An unpaired t-test was performed. Error bars represent the mean ± S.E.M. ** p < 0.005 ( D , E ) Western blot analysis of harvested tumor tissue was performed after the conclusion of the dosing experiment. Both p53 and xCT protein levels were decreased in PRIMA-1 Met -treated animals compared to saline controls. An unpaired, one-tailed t-test was performed. ** p < 0.01, * p < 0.05.

Techniques: Mutagenesis, In Vivo, Tumor Implantation, Saline, Western Blot, One-tailed Test

$Growth kinetics, cell cycle checkpoint control, and suppression of recombination of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. A. Representative growth kinetics of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. 750,000 cells were plated onto 10cm dishes and collected and counted on the days indicated. T-tests on multiple growth experiments indicated no significant difference between p53+/+ and p53+/m growth rates (P = 0.61), while growth rate differences between p53+/m and p53+/− MEFs were significantly different (P = 0.003). B. S phase fractions in rapidly dividing MEFs of different p53 genotypes. MEFs were analyzed for DNA content and BrdU incorporation by flow cytometry and the percentage of cells in S phase was calculated. T-tests indicated the S phase fractions of p53+/m and p53+/+ MEFs were not significantly different (P = 0.47), while S phase fractions of p53+/m and p53+/− MEFs were significantly different and are indicated by an asterisk (P = 0.05). C. Suppression of recombination in p53+/+, p53+/−, p53+/m, and p53−/− passage 1−3 MEFs. p53+/+, p53+/−, p53+/m, and p53−/− MEFs were infected with a retrovirus expressing two tandem copies of mutant forms of a GFP-Zeocin gene as well as a neomycin resistance marker. MEFs were selected with G418 or G418 plus Zeocin to determine the recombination frequencies. The p53+/m MEFs have a recombination frequency roughly equal to that of p53+/+ MEFs. The difference in recombination frequency between p53+/m and p53+/− MEFs is highly significant (P < .0001) and is indicated by three asterisks. Differences between p53+/+ and p53+/− MEFs are also statistically significant (P < .0001).$

Journal:

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

doi: 10.1016/j.mad.2007.10.011

Figure Lengend Snippet: Growth kinetics, cell cycle checkpoint control, and suppression of recombination of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. A. Representative growth kinetics of p53+/+, p53+/−, p53+/m, and p53−/− MEFs. 750,000 cells were plated onto 10cm dishes and collected and counted on the days indicated. T-tests on multiple growth experiments indicated no significant difference between p53+/+ and p53+/m growth rates (P = 0.61), while growth rate differences between p53+/m and p53+/− MEFs were significantly different (P = 0.003). B. S phase fractions in rapidly dividing MEFs of different p53 genotypes. MEFs were analyzed for DNA content and BrdU incorporation by flow cytometry and the percentage of cells in S phase was calculated. T-tests indicated the S phase fractions of p53+/m and p53+/+ MEFs were not significantly different (P = 0.47), while S phase fractions of p53+/m and p53+/− MEFs were significantly different and are indicated by an asterisk (P = 0.05). C. Suppression of recombination in p53+/+, p53+/−, p53+/m, and p53−/− passage 1−3 MEFs. p53+/+, p53+/−, p53+/m, and p53−/− MEFs were infected with a retrovirus expressing two tandem copies of mutant forms of a GFP-Zeocin gene as well as a neomycin resistance marker. MEFs were selected with G418 or G418 plus Zeocin to determine the recombination frequencies. The p53+/m MEFs have a recombination frequency roughly equal to that of p53+/+ MEFs. The difference in recombination frequency between p53+/m and p53+/− MEFs is highly significant (P < .0001) and is indicated by three asterisks. Differences between p53+/+ and p53+/− MEFs are also statistically significant (P < .0001).

Article Snippet: Membranes were probed with p53 antibodies (FL393 or DO-1, Santa Cruz) or GFP (B-2, Santa Cruz) to detect GFP tagged p53 and M. To detect MDM2, membranes were probed with a mixture of Ab-4 and Ab-5 (Calbiochem).

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

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

Journal:

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

doi: 10.1016/j.mad.2007.10.011

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

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

The M protein localizes to the nucleus and interacts with wild-type p53. A. Diagram of human p53 and the M protein. Residues that were mutated to alanine in the NLS and tetramerization domain are shown in bold. AD: Activation Domain. DBD: DNA Binding Domain. NLS/TD: Nuclear Localization Signal/Tetramerization Domain. B. Localization of M and M mutants. Cells were transfected with GFP tagged M (panels a-c), GFP tagged MKRKKK (panels d-f), or GFP tagged M348/350A (panels g-i) as indicated. The localization of M was determined by immunofluorescence and nuclei visualized by DAPI staining. 300 cells were counted and scored as having GFP-M mostly nuclear (N), mostly cytoplasmic (C), or both nuclear and cytoplasmic (B). Percentages listed are an average of three independent experiments. C. Interaction between p53 and M mutants. p53−/− HCT116 cells were transfected with GFP tagged p53 and M expression constructs as indicated. p53 was immunoprecipitated with an antibody specific to the N-terminus of p53. Membranes were probed with an antibody to GFP to detect both full-length p53 and the M protein.

Journal:

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

doi: 10.1016/j.mad.2007.10.011

Figure Lengend Snippet: The M protein localizes to the nucleus and interacts with wild-type p53. A. Diagram of human p53 and the M protein. Residues that were mutated to alanine in the NLS and tetramerization domain are shown in bold. AD: Activation Domain. DBD: DNA Binding Domain. NLS/TD: Nuclear Localization Signal/Tetramerization Domain. B. Localization of M and M mutants. Cells were transfected with GFP tagged M (panels a-c), GFP tagged MKRKKK (panels d-f), or GFP tagged M348/350A (panels g-i) as indicated. The localization of M was determined by immunofluorescence and nuclei visualized by DAPI staining. 300 cells were counted and scored as having GFP-M mostly nuclear (N), mostly cytoplasmic (C), or both nuclear and cytoplasmic (B). Percentages listed are an average of three independent experiments. C. Interaction between p53 and M mutants. p53−/− HCT116 cells were transfected with GFP tagged p53 and M expression constructs as indicated. p53 was immunoprecipitated with an antibody specific to the N-terminus of p53. Membranes were probed with an antibody to GFP to detect both full-length p53 and the M protein.

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

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

Journal:

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

doi: 10.1016/j.mad.2007.10.011

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

Techniques: Transfection, Plasmid Preparation, Irradiation, Immunostaining

M induces nuclear localization of a p53 NLS mutant (p53KRKKK) and this is dependent on the nuclear localization signal of M and efficient interaction between p53 and the M protein. A. p53−/− HCT116 cells were transfected with the plasmids indicated. Localization of full-length p53 was determined by immunofluorescence. B. Quantification of immunofluorescence staining in A. 300 cells per transfection condition were scored as having primarily nuclear p53, primarily cytoplasmic p53, or both nuclear and cytoplasmic p53. Values are the average of three independent experiments and error bars represent standard error. * p<.001; p53KRKKK is compared to p53wt and all others are compared to p53KRKKK.

Journal:

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

doi: 10.1016/j.mad.2007.10.011

Figure Lengend Snippet: M induces nuclear localization of a p53 NLS mutant (p53KRKKK) and this is dependent on the nuclear localization signal of M and efficient interaction between p53 and the M protein. A. p53−/− HCT116 cells were transfected with the plasmids indicated. Localization of full-length p53 was determined by immunofluorescence. B. Quantification of immunofluorescence staining in A. 300 cells per transfection condition were scored as having primarily nuclear p53, primarily cytoplasmic p53, or both nuclear and cytoplasmic p53. Values are the average of three independent experiments and error bars represent standard error. * p<.001; p53KRKKK is compared to p53wt and all others are compared to p53KRKKK.

Techniques: Mutagenesis, Transfection, Immunofluorescence, Staining

p53 protein levels are more stable in p53+/m MEFs compared to p53+/− MEFs. A. p53 protein levels in the absence of de novo protein synthesis. Low passage MEFs were treated with cycloheximide and cell lysates collected at the indicated time points. B. p53+/m MEFs exhibit increased p53 stability compared to p53+/− MEFs. Western blots were normalized to actin, quantitated by densitometry and graphed as the percent p53 remaining over time relative to the zero time point. The graph represents the average of three independent experiments (and independent MEF lines). Error bars represent standard error of the mean. Statistical analyses (ANOVA) indicated that cycloheximide treated p53+/m cells exhibited significant increases in p53 stability compared to similarly treated p53+/− cells (P = 0.04). C. p53 protein levels in p53+/m and p53+/− MEFs after treatment with proteasome inhibitors. Two different isolates of of low passage p53+/− MEFs (MEF-1: lanes 1,2; MEF-2: lanes 3,4) and p53+/m MEFs (MEF-3: lanes 5,6; MEF-4: lanes 7,8) were untreated or treated with proteasome inhibitors MG101 and MG132 for 6 hours prior to harvest. p53 protein levels accumulate upon proteasome inhibitor treatment in p53+/− MEFs, but are not significantly increased in p53+/m MEFs implying that p53 is protected from proteasomal degradation in p53+/m MEFs. D. p53 protein levels are less sensitive to proteasome inhibitors in p53+/m MEFs. Western blots in C were normalized to actin and quantitated by densitometry. The values shown are the average p53 levels of three independent MEF lines for each genotype and error bars represent standard error. *P < 0.01 compared to p53+/− untreated control. CHX: cycloheximide.

Journal:

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

doi: 10.1016/j.mad.2007.10.011

Figure Lengend Snippet: p53 protein levels are more stable in p53+/m MEFs compared to p53+/− MEFs. A. p53 protein levels in the absence of de novo protein synthesis. Low passage MEFs were treated with cycloheximide and cell lysates collected at the indicated time points. B. p53+/m MEFs exhibit increased p53 stability compared to p53+/− MEFs. Western blots were normalized to actin, quantitated by densitometry and graphed as the percent p53 remaining over time relative to the zero time point. The graph represents the average of three independent experiments (and independent MEF lines). Error bars represent standard error of the mean. Statistical analyses (ANOVA) indicated that cycloheximide treated p53+/m cells exhibited significant increases in p53 stability compared to similarly treated p53+/− cells (P = 0.04). C. p53 protein levels in p53+/m and p53+/− MEFs after treatment with proteasome inhibitors. Two different isolates of of low passage p53+/− MEFs (MEF-1: lanes 1,2; MEF-2: lanes 3,4) and p53+/m MEFs (MEF-3: lanes 5,6; MEF-4: lanes 7,8) were untreated or treated with proteasome inhibitors MG101 and MG132 for 6 hours prior to harvest. p53 protein levels accumulate upon proteasome inhibitor treatment in p53+/− MEFs, but are not significantly increased in p53+/m MEFs implying that p53 is protected from proteasomal degradation in p53+/m MEFs. D. p53 protein levels are less sensitive to proteasome inhibitors in p53+/m MEFs. Western blots in C were normalized to actin and quantitated by densitometry. The values shown are the average p53 levels of three independent MEF lines for each genotype and error bars represent standard error. *P < 0.01 compared to p53+/− untreated control. CHX: cycloheximide.

Techniques: Western Blot, Control

The M protein enhances the stability of full-length p53. U2OS cells were transfected with plasmids encoding M and M mutants as indicated. A. p53 protein levels in the absence of de novo protein synthesis. Cells were treated with cycloheximide and protein lysates collected at the indicated time points followed by a Western blot for total p53. B. Graphical representation of total p53 levels in A after normalization to actin levels. Blots were scanned on a GE Storm 860 imager and quantitated using ImageQuant software. The graphs indicate the percent p53 remaining relative to the zero time point. Values represent the average of three independent experiments and error bars represent standard error. Statistical analyses (ANOVA) indicated that empty vector transfected U2OS cells exhibited significantly less p53 stability than cells transfected with M or mutant M vectors (P < .0001). C. p53 protein levels after treatment with proteasome inhibitors. Following transfection with empty vector or M, U2OS cells were treated with the proteasome inhibitors MG101 and MG132 for 6 hours. Cells were harvested and p53 protein levels analyzed by Western blot. p53 accumulates in the presence of the proteasome inhibitors in cells transfected with empty vector. However, p53 protein levels are not affected in cells transfected with M, indicating that the M protein protects p53 from proteasomal degradation.

Journal:

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

doi: 10.1016/j.mad.2007.10.011

Figure Lengend Snippet: The M protein enhances the stability of full-length p53. U2OS cells were transfected with plasmids encoding M and M mutants as indicated. A. p53 protein levels in the absence of de novo protein synthesis. Cells were treated with cycloheximide and protein lysates collected at the indicated time points followed by a Western blot for total p53. B. Graphical representation of total p53 levels in A after normalization to actin levels. Blots were scanned on a GE Storm 860 imager and quantitated using ImageQuant software. The graphs indicate the percent p53 remaining relative to the zero time point. Values represent the average of three independent experiments and error bars represent standard error. Statistical analyses (ANOVA) indicated that empty vector transfected U2OS cells exhibited significantly less p53 stability than cells transfected with M or mutant M vectors (P < .0001). C. p53 protein levels after treatment with proteasome inhibitors. Following transfection with empty vector or M, U2OS cells were treated with the proteasome inhibitors MG101 and MG132 for 6 hours. Cells were harvested and p53 protein levels analyzed by Western blot. p53 accumulates in the presence of the proteasome inhibitors in cells transfected with empty vector. However, p53 protein levels are not affected in cells transfected with M, indicating that the M protein protects p53 from proteasomal degradation.

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

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

Journal:

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

doi: 10.1016/j.mad.2007.10.011

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

Techniques: Transfection, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

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1) Product Images from "Specific TP53 mutations impair the recruitment of 53BP1 to DNA double-strand breaks underlying the mechanism of radioresistance"

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