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

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: 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

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: Clonogenic Cell Survival Assay, Mutagenesis, Irradiation, Immunofluorescence

Journal: Gene

Article Title: An arrayed human genomic library constructed in the PAC shuttle vector pJCPAC-Mam2 for genome-wide association studies and gene therapy

doi: 10.1016/j.gene.2012.01.011

Figure Lengend Snippet: Live-cell imaging of p53-GFP-expressing Saos-2 cells. Saos-2 cells were transfected as described previously with pJCPAC-Mam1-p53-GFP. Three days post-transfection, Saos-2 cells were photographed and UV and white light images were merged. (A). Exogenous p53-GFP localizes predominantly to the nuclei of Saos-2 cells. (B). Membrane blebbing, a hallmark event in apoptosis occurs in a subset of p53-GFP-expressing Saos-2 cells.

Article Snippet: A p53-GFP cassette (Gift of Jon Jarvik, SpectraGenetics) was subcloned into the unique NotI site of pJCPAC-Mam1 and Saos-2 cells were also transfected with this p53-GFP fusion protein-expressing construct using PEI as previously described.

Techniques: Live Cell Imaging, Expressing, Transfection

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A, B ) p53 −/− kidneys exhibit 50% fewer and less complex LTA-positive proximal tubules. Metanephroi were harvested at E11.5, cultured on trans-well filters for 72 h and stained for cytokeratin and LTA. LTA counts were averaged from kidneys collected from at least 4 embryos. Mutant kidneys exhibit significantly fewer LTA+ structures than wild-type kidneys (p<0.005). C) E17.5 kidneys were harvested from embryos from p53 +/− crosses, formalin-fixed and sectioned for immunostaining (Methods). Sections were stained with WT1 and cytokeratin antibodies. P53 −/− kidneys show paucity of WT1 stained nephrons. D ) Kidneys from mice with conditional p53 deletion from Six2+ cap mesenchyme (CM p53−/− ). Hypoplasia persists post-natally, shown in P0 kidneys, top panels at 4x. Bottom panels (x10) show fewer Lhx1-positive nascent nephrons (red). At P0, 6/8 (75%) examined CM p53−/− kidneys were hypoplastic compared to wild-type littermate kidneys (n = 8).

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Cell Culture, Staining, Mutagenesis, Immunostaining

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) p53 expression overlaps that of Pax2 in the developing kidney. Immunofluorescence staining was done on E15.5 kidney sections to visualize Pax2 and p53 protein expression. p53 co- localizes with Pax2 in the cap mesenchyme (CM) and in the ureteric tip (UB). B ) In situ hybridization. Decreased expression of Pax2 mRNA at E12.5. C ) QPCR. Pax2 mRNA is significantly lower in germ-line p53 −/− kidneys. QPCR was done on RNA from individual E15.5 kidney pairs from wild-type and p53 −/− embryos (n = 4). D ) Pax2 protein in E15.5 kidneys, detected by IF staining. Both wild-type and mutant kidney sections were processed for IF staining simultaneously and images captured at identical exposure setting. After normalizing intensity to that in wild-type, images were converted to a heat-map to demonstrate differential Pax2 staining between wild-type and mutant kidneys. High intensity staining corresponds to red/yellow areas and lower intensity corresponds to green/blue. Top panels at x4 and bottom panels at x20. E ) Quantitation of staining intensity was done using Slidebook software and is shown graphically. UB, ureteric bud/tips; CM, cap mesenchyme; Nn, nascent nephrons including pretubular aggregates and renal vesicles.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Expressing, Immunofluorescence, Staining, In Situ Hybridization, Mutagenesis, Quantitation Assay, Software

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) Reduced Pax2 staining in the CM of E12.5 CM p53−/− kidneys in comparison to wild-type CM, indicative of lower Pax2 protein levels. B ) Pax2 mRNA is ∼40% lower in CM p53−/− kidneys. QPCR was done on RNA from E15.5 littermate kidneys from wild-type (n = 5 pairs) and CM p53−/− (n = 5 pairs).

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Staining, Comparison

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: ) Genomic view of p53 occupancy at the Pax2 gene locus identified by ChIP-Seq and visualized using the Integrated Genome Browser (IGB). Orange bars denote p53 enriched regions after background (Input) subtraction. Green bars show MACS peaks which represent points of highest density of sequenced fragments within the orange interval. Peak 1 and 2 are in the proximal promoter and in intron2-exon3, respectively. Two additional peaks were identified far distal to the Pax2 gene at ∼−14 kb (peak 3) and ∼−83 kb (peak 4) from the transcription start site. Yellow box shows region validated for p53-enrichment by ChIP-PCR in Fig. 5. B ) High sequence conservation in mammals of regions encompassing p53-enriched areas, including the non-genic distal regions 3 and 4, visualized in the UCSC genome browser ( http://genome.ucsc.edu ). C ) Red bars show location of p53 binding motifs in and around p53-occupied regions 1 and 2 in the Pax2 promoter/gene. Although p53 binding sites are broadly scattered across the entire region including the intervening region between regions 1 and 2 (see ), p53 occupancy occurs at specific regions.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: ChIP-sequencing, Sequencing, Binding Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: ) (a) ChIP-Seq track showing p53 occupancy at Pax2 proximal promoter (Region 1). (b) That this enrichment is not an artifact of amplification and sequencing is shown by lack of enrichment in the Input sample. Presence of any spurious false peaks in the Input sample eliminates that region from an interval, reflecting high stringency set for the MACS program. (c) Grey bar shows region of p53 enrichment designated as an interval. The dotted green arrow denotes RefSeq TSS, solid green arrow shows TSS described by . (d) p53 motifs (blue bars) identified by Genomatix and manually. (e) Position of amplicons to validate ChIP-Seq data and show differential p53 occupancy between mK3 and mK4 cells by ChIP-PCR. (f) Co-ordinates on mus chromosome 19. B ) Chromatin was immunoprecipitated from both cell lines with anti-p53 antibody and amplified by PCR. PCR band intensities were quantified using the Alphaimager software as described in , and band intensity of immunoprecipitated fragments was normalized to Input band intensity for each cell line. Normalized values for each amplicon were plotted as mK4/mK3 ratios. Values greater than 1.0 indicate fragment enrichment in mK4 relative to mK3, as a result of increased p53 binding and immunoprecipitation as seen for amplicons P3 and P7 (blue line). Values are a mean of three independent ChIP experiments.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: ChIP-sequencing, Amplification, Sequencing, Immunoprecipitation, Software, Binding Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) p53 −/− H1299 cells were co-transfected with a p53 expression plasmid (pCMV-p53) and a 4.1-Pax2-CAT reporter construct. Dose-dependent increase in reporter activity was observed with wild-type p53 (WT-p53). Reporter activity was maintained at baseline when reporter plasmid was co-transfected with a non-DNA binding mutant p53 (Mt-p53, pCMVp53-E258K). This mutant acts as a dominant-negative and inhibits Pax2 promoter activation by wild-type p53. B ) mK4 cells were transiently co-transfected with a p53 expression plasmid (pCMV-p53) and a 4.1-Pax2-Luc reporter construct. Absolute values of luciferase activity are plotted as Relative Light Units (RLU). Luciferase activity was normalized to protein concentration. C ) p53 knock-down in mK4 cells using 4 different p53 shRNA plasmids showed corresponding decrease in Pax2 mRNA, relative to mRNA levels from GFP- cells (dotted red line). mK4 cells were transfected with p53-shRNA-GFP plasmid, and GFP+ cells were sorted 48 h post-transfection, RNA harvested and used for QPCR. Gene expression was normalized to β-actin or GAPDH expression. D ) Schematic of deletion mutant Pax2 promoter constructs, shown with respect to p53 occupancy determined by ChIP-Seq. Orange bar denotes p53 occupancy in Pax2 promoter, corresponding to region 1 in Fig. 2A. Green solid arrow shows TSS used in this study, dotted arrow denotes possible alternate TSS in RefSeq. Purple bars indicate location of p53 binding sites identified in silico and tested for p53 binding by EMSA, numbered 1–14. Mutagenized sites shown in red. E ) Full-length or truncated Pax2-promoter-CAT reporter plasmids were transfected into UB cells either without or with pCMV-p53 (50 ng). CAT activity was normalized to protein concentration. Fold-induction by p53 of CAT activity over baseline (red-line) is plotted. F ) Fold-change in reporter activity by p53 after site-directed mutagenesis of p53-binding site 7 (Mut 7–1 and 7–2) and 8 (Mut 8–1 and 8–2) individually or together (Mut 7/8). Two clones were tested in transient transfection assays per site mutated.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Activity Assay, Binding Assay, Mutagenesis, Dominant Negative Mutation, Activation Assay, Luciferase, Protein Concentration, Knockdown, shRNA, Gene Expression, ChIP-sequencing, In Silico, Clone Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: Percent conservation in mammals of p53 binding sites in the Pax2 promoter.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A ) E13.5 kidneys from Pax2 LacZ/+; p53 +/− crosses were stained with cytokeratin and UB tips counted. B ) UB tip number was used as a phenotypic readout and measure of renal hypoplasia in Pax2 LacZ/+; p53 −/− animals compared to wild type, or Pax2 LacZ/+ ;p53 +/+ . Reduction of either Pax2 gene dosage or elimination of p53 gene reduced UB tip number by ∼12–15%. Complete p53 deficiency with Pax2 haploinsufficiency resulted in ∼55% decrease in tip number. For each genotype, N = 3−5 kidney pairs from at least 3 independent litters. All animals were harvested at ∼E13.0, cultured overnight and fixed for whole mount cytokeratin staining to enable counting of UB tips.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Staining, Cell Culture

Journal: PLoS ONE

Article Title: A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis

doi: 10.1371/journal.pone.0044869

Figure Lengend Snippet: A) Model for autonomous and interactive gene regulation by p53 and Pax2. Genes regulated independently by each transcription factor. P53 positively regulates Pax2 expression, and the two may co-operatively regulate genes shown in box below dotted arrow. Expression of these genes is altered in p53 −/− E15.5 kidneys and these genes contain p53-binding motifs that are occupied by p53 in vivo as determined by ChIP-Seq. Genomatix search revealed Pax2 binding motifs in these genes. B) Model for p53-Pax2 cross-talk in the developing kidney. p53 enhances Pax2 expression in mesenchyme cells and promotes their transition to epithelia. Thus, p53 serves to promote differentiation of progenitor cells to nascent nephrons, and in its absence Pax2 down-regulation is a possible mechanism of nephron deficit observed in these kidneys.

Article Snippet: mK4 cells were transfected with p53-shRNA-GFP plasmid (SuperArray Biosciences, KM0931G) for 72 h. Transfected cells were sorted by FACS and RNA was isolated from GFP + and GFP - cells.

Techniques: Expressing, Binding Assay, In Vivo, ChIP-sequencing