Journal: Nature Communications

Article Title: TP53 variant clusters stratify phenotypic diversity in germline carriers and reveal an osteosarcoma-prone subgroup

doi: 10.1038/s41467-025-63528-6

Figure Lengend Snippet: a Matrix correlation plots comparing scaled data (Z-score normalization) TP53 mutagenesis datasets covering the entire protein (LOF loss-of-function, DDR DNA damage repair, DN dominant negative activity, TA transcriptional activity) , . Variants are colour-coded based on the functional domain they reside in: NTD N-terminal domain (red), DBD DNA-binding domain (black), OD oligomerization domain (cyan), CTD C-terminal domain (purple). Linear relationships between functional screens were assessed using Pearson’s correlation coefficient (r). b Principal component analysis (PCA) and unsupervised k-means clustering performed with TP53 mutagenesis cellular functional assay measurements. c Uniform manifold approximation and projection (UMAP) performed using TP53 mutagenesis cellular functional assay measurements and colour-coded based on PCA k-means clustering ( N = 200, D = 0.4). d Heatmap displaying the codon frequencies and distributions of TP53 variant clusters. Red arrowheads indicate variant hotspots in cluster 5. PRR = proline-rich region. e –h Violin plots comparing the functional consequences of variants within each cluster. Sample sizes: cluster 1 ( n = 791), cluster 2 ( n = 419), cluster 3 ( n = 381), cluster 4 ( n = 448), cluster 5 ( n = 269). P -values on plots were calculated using Kruskal-Wallis tests. Two-tailed Mann-Whitney U tests were used for pairwise comparisons (**** p < 0.0001). Source data are provided as a Source Data file.

Article Snippet: TP53 and β-actin were detected using a mouse anti-human TP53 monoclonal antibody (mAb; 1:1000 dilution; IC12, Cell Signalling) and rabbit anti-human β-actin (1:1000 dilution; 13E5, Cell Signalling).

Techniques: Mutagenesis, Dominant Negative Mutation, Activity Assay, Functional Assay, Binding Assay, Variant Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: TP53 variant clusters stratify phenotypic diversity in germline carriers and reveal an osteosarcoma-prone subgroup

doi: 10.1038/s41467-025-63528-6

Figure Lengend Snippet: a Stacked bar plot showing VCEP annotated variants applied to the variant clusters ( n = 106). Cluster 1 (42 classified of 791), cluster 2 (18 classified of 419), cluster 3 (21 classified of 381), cluster 4 (22 classified of 448), cluster 5 (3 classified of 269). b Bar graphs displaying the frequencies of germline variants in each cluster found in the NCI TP53 Database ( n = 3113), ( c ) a multi-institutional validation cohort ( n = 458), ( d ) gnomAD non-cancer dataset ( n = 186), and ( e ) FLOSSIES database ( n = 27). P -values on plots were calculated using chi-square tests. Source data are provided as a Source Data file.

Article Snippet: TP53 and β-actin were detected using a mouse anti-human TP53 monoclonal antibody (mAb; 1:1000 dilution; IC12, Cell Signalling) and rabbit anti-human β-actin (1:1000 dilution; 13E5, Cell Signalling).

Techniques: Variant Assay, Biomarker Discovery

Journal: Nature Communications

Article Title: TP53 variant clusters stratify phenotypic diversity in germline carriers and reveal an osteosarcoma-prone subgroup

doi: 10.1038/s41467-025-63528-6

Figure Lengend Snippet: a Line plot displaying the cumulative cancer diagnosis ages over time and a box-and-whisker plot representing the ages at cancer diagnosis in the NCI TP53 Database (**** p < 0.0001 compared to clusters 1 and 4, * p = 0.014 vs cluster 1, * p = 0.024 vs cluster 4); sample sizes: cluster 1 ( n = 81), cluster 2 ( n = 181), cluster 3 ( n = 1126), cluster 4 ( n = 46), cluster 5 ( n = 40), FS/NS/DEL ( n = 407). b Line plot displaying the cumulative cancer diagnosis ages over time and a box-and-whisker plot representing the ages at cancer diagnosis in the validation cohort (* p < 0.0001 compared to clusters 3 and FS/NS/DEL, *p = 0.014 compared to cluster 2, * p = 0.022 vs cluster 5, * p = 0.054 vs cluster 4); sample sizes: cluster 1 ( n = 13), cluster 2 ( n = 86), cluster 3 ( n = 255), cluster 4 ( n = 3), cluster 5 ( n = 4), FS/NS/DEL ( n = 125). c Line plot displaying the frequency of breast tumor diagnosis over time and a box-and-whisker plot representing the ages at cancer diagnosis (*** p = 0.0003 vs cluster 1, *** p < 0.0001 vs cluster 2, *** p = 0.0002 vs cluster 4, ** p = 0.0042 vs cluster 1, ** p = 0.0064 vs cluster 2, ** p = 0.0013 vs cluster 4, **** p < 0.0001 vs clusters 1, 2, and 4); sample sizes: cluster 1 ( n = 15), cluster 2 ( n = 69), cluster 3 ( n = 307), cluster 4 ( n = 15), cluster 5 ( n = 13), FS/NS/DEL ( n = 143). d Line plot displaying the frequency of ACC diagnosis over time and box-and-whisker plot representing the age at ACC onset (** p = 0.0045 vs cluster 2, ** p = 0.0002 vs cluster 3, ** p < 0.0001 vs FS/NS/DEL); sample sizes: cluster 1 ( n = 2), cluster 2 ( n = 18), cluster 3 ( n = 73), cluster 4 ( n = 4), cluster 5 ( n = 2), FS/NS/DEL ( n = 21). For all box-and-whisker plots, the center line represents the median, the box spans the interquartile range (25 th to 75 th percentiles), and the whiskers extend to the 10 th and 90 th percentiles. P-values on plots were calculated using Kruskal-Wallis tests. Two-tailed Mann-Whitney U tests were used for pairwise comparisons. Source data are provided as a Source Data file.

Article Snippet: TP53 and β-actin were detected using a mouse anti-human TP53 monoclonal antibody (mAb; 1:1000 dilution; IC12, Cell Signalling) and rabbit anti-human β-actin (1:1000 dilution; 13E5, Cell Signalling).

Techniques: Biomarker Discovery, Whisker Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: TP53 variant clusters stratify phenotypic diversity in germline carriers and reveal an osteosarcoma-prone subgroup

doi: 10.1038/s41467-025-63528-6

Figure Lengend Snippet: a Stacked bar plot displaying the cancer type distribution patterns manifested in germline TP53 variant carriers. Cluster 1 ( n = 105), cluster 2 ( n = 219), cluster 3 ( n = 1255), cluster 4 ( n = 55), cluster 5 ( n = 50), FS/NS/DEL ( n = 467). b Bar graph showing the frequency of LFS-core cancer types (** FDR = 0.0035 vs cluster 2 and 3, ** FDR = 0.0012 vs FS/NS/DEL). c Bar graph showing the frequency of colorectal cancers (* FDR = 0.0092 vs cluster 2, * FDR = 0.00015 vs cluster 3 and FS/NS/DEL). d Bar graph showing the frequency of patients experiencing multiple tumors (** FDR = 0.0031 cluster 3 vs cluster 1, ** FDR = 0.0002 cluster 3 vs cluster 2, ** FDR = 0.0031 cluster 3 vs cluster 4, ** FDR = 0.0097 FS/NS/DEL vs cluster 1, ** FDR = 0.0031 FS/NS/DEL vs cluster 2, ** FDR = 0.0073 FS/NS/DEL vs cluster 4). e Bar graph showing frequency of carriers fulfilling classic LFS or Chompret criteria (**** FDR < 0.0001 comparing to clusters 3, 5, and FS/NS/DEL). f Bar graph showing frequency of carriers fulfilling LFS-like criteria (LFL; Eeles and Birch criteria , ) (** FDR = 0.0027 vs cluster 1, ** FDR < 0.0001 vs cluster 2, ** FDR = 0.0007 vs cluster 3, ** FDR = 0.0005 vs cluster 5, ** FDR < 0.0001 vs FS/NS/DEL). g Bar graph showing the frequency of osteosarcomas (** FDR = 0.0003 vs cluster 1, ** FDR = 0.0002 vs cluster 2, ** FDR = 0.0013 vs cluster 3, ** FDR = 0.0003 vs cluster 4, ** FDR = 0.004 vs FS/NS/DEL). P -values on plots were calculated using chi-square tests. Two-tailed Fisher’s exact tests were used for pairwise comparisons with FDR -corrected p-values. Source data are provided as a Source Data file.

Article Snippet: TP53 and β-actin were detected using a mouse anti-human TP53 monoclonal antibody (mAb; 1:1000 dilution; IC12, Cell Signalling) and rabbit anti-human β-actin (1:1000 dilution; 13E5, Cell Signalling).

Techniques: Variant Assay, Two Tailed Test

Journal: Nature Communications

Article Title: TP53 variant clusters stratify phenotypic diversity in germline carriers and reveal an osteosarcoma-prone subgroup

doi: 10.1038/s41467-025-63528-6

Figure Lengend Snippet: a Bar graphs displaying clonogenic assay results in Saos-2 p53-null osteosarcoma cells and ( b ) in H1299 p53-null lung adenocarcinoma cells expressing TP53 variants representing each cluster (mean with SEM). c Bar graph showing clonogenic assay results in H1299 cells comparing R175 variants (mean with SEM). d Box-and-whisker plot of metabolic growth rates as measured by the MTT colorimetric assay using unstressed dermal fibroblasts from patient skin biopsies. e Box-and-whisker plot of TP53 variant scores as measured by mean TP53 target gene mRNA expression in patient blood-derived cells (fresh peripheral blood lymphocytes or lymphoblastoid cell lines) . Wild-type (WT; n = 54), cluster 1 ( n = 7), cluster 2 ( n = 8), cluster 3 ( n = 19), cluster 4 ( n = 4), FS/NS/DEL ( n = 8). Clonogenic and MTT assays were repeated at least twice and performed in triplicate and sextuplicate, respectively. For all box-and-whisker plots, the center line represents the median, the box spans the interquartile range (25 th and 75 th percentiles), and the whiskers extend to the minimum and maximum values. All pairwise statistical comparisons in the clonogenic assay are made relative to WT p53. P -values were calculated using one-tailed unpaired t-tests. Exact p -values for all pairwise comparisons are provided in Supplementary Data . Source data are provided as a Source Data file.

Article Snippet: TP53 and β-actin were detected using a mouse anti-human TP53 monoclonal antibody (mAb; 1:1000 dilution; IC12, Cell Signalling) and rabbit anti-human β-actin (1:1000 dilution; 13E5, Cell Signalling).

Techniques: Clonogenic Assay, Expressing, Whisker Assay, Colorimetric Assay, Variant Assay, Derivative Assay, One-tailed Test

Journal: Nature Communications

Article Title: TP53 variant clusters stratify phenotypic diversity in germline carriers and reveal an osteosarcoma-prone subgroup

doi: 10.1038/s41467-025-63528-6

Figure Lengend Snippet: This schematic representation illustrates TP53 variant clusters positioned along a continuum of pathogenicity, ordered by increasing cancer risk. WT (teal) corresponds to normal p53 function and no elevated cancer risk. Cluster 1 (red) includes benign or conditionally pathogenic variants associated with lower risk. Cluster 4 (blue) comprises variants that retain WT-like function but are linked to attenuated phenotypes and intermediate risk. Cluster 2 (olive green) includes hypomorphic or partially functional variants that confer intermediate risk. Cluster 5 (magenta) encompasses monomeric variants with high risk as well as others with unclear mechanisms and uncertain risk. T (grey) represents truncating variants associated with high risk due to loss of p53 activity. Cluster 3 (green) consists of DN variants that interfere with WT function and confer high risk.

Article Snippet: TP53 and β-actin were detected using a mouse anti-human TP53 monoclonal antibody (mAb; 1:1000 dilution; IC12, Cell Signalling) and rabbit anti-human β-actin (1:1000 dilution; 13E5, Cell Signalling).

Techniques: Variant Assay, Functional Assay, Activity Assay

Journal: Virology

Article Title: The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8.

doi: 10.1016/j.virol.2013.12.013

Figure Lengend Snippet: Fig. 4. HPV-18 E6 induces active caspase 8 expression in the nucleus of HeLa cells. Panel A. Cells were transfected with siRNA luciferase (L) or siRNA E6/E7 (E6) and after 72 h the cells were harvested and fractionated into cytoplasmic (I), membrane (II), nuclear (III) and cytoskeletal pools (IV). The fractions were then subjected to Western blot analysis for caspase 8 (p57 full length and active p18) and p53. Markers for loading and fraction integrity were vimentin, tubulin, p84 and transferrin receptor (TR). Panel B. HeLa cells were transfected with siRNA luciferase (si luc) or siRNA E6/E7 (si E6) and after 72 h cells were fixed and stained for p53 and caspase 8. HaCaT cells were included for comparison. Note the strong increase in nuclear p53 following E6/E7 ablation and the corresponding increase in cytoplasmic caspase 8, which is indicated by the arrows.

Article Snippet: HeLa and HaCaT cells were processed in the same manner 72 h post siRNA transfection and were incubated with anti p53 (Santa Cruz) and anti-caspase 8 antibody (IC12, Cell Signalling).

Techniques: Expressing, Transfection, Luciferase, Membrane, Western Blot, Staining, Comparison

Journal: Cell death and differentiation

Article Title: Defective MHC class I antigen surface expression promotes cellular survival through elevated ER stress and modulation of p53 function.

doi: 10.1038/cdd.2008.55

Figure Lengend Snippet: Figure 5 Lack of MHC class I expression in TAP-deficient RMAS cells also lead to enhanced resistance to apoptosis and elevated ER stress. (a) Transporter associated with antigen processing (TAP) proficient (RMA) and deficient (RMAS) isogenic cells were stained with anti-H-2kb and H-2kk antibodies. RMAS cells have markedly reduced expression of H-2kb. (b) RMA and RMAS cells were treated with cisplatin (50 mM) or UV (50 J/m2) or g- irradiated (20 Gy), and cell death was determined after 20 h, as described. Percentage dead cells are indicated. (c) Immunoblot analysis of p53 levels after g-irradiation (20 Gy, 16 h) of RMA and RMAS cells is shown. (d) Reverse transcriptase-PCR (RT-PCR; left panel) and immunoblot (right panel) analysis of XBP-1 was performed using RMA and RMAS cells, as described above

Article Snippet: The following antibodies were used for immunoblot analysis: p53 (CM5 – Novocastra) and (IC12 – Cell Signaling), phospho-ser18, -ser 315, and -389 p53, GSK-3b (Cell Signaling), phospho-PERK (US Biologicals), MDM2, Puma, Cyclin G, XBP-1, PERK, p21, Tubulin (Santa Cruz), Lamin (Cell Signaling), and Actin (Sigma).

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

Journal: International journal of nephrology

Article Title: Constitutional Nephrin Deficiency in Conditionally Immortalized Human Podocytes Induced Epithelial-Mesenchymal Transition, Supported by β-Catenin/NF-kappa B Activation: A Consequence of Cell Junction Impairment?

doi: 10.1155/2013/457490

Figure Lengend Snippet: Figure 5: 𝛽-Catenin, TCF-4, LEF-1, p53, and pRB distribution and expression in wild type and nephrin-mutated podocytes. The cells were lysated, fractionated and processed for western blot as described in Section 2. (a) Distribution and expression of 𝛽-catenin in cell membranes and nuclear compartment. The goat anti-𝛽-catenin (sc-1496) antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). (b) Expression of LEF-1 and TCF-4 in nuclear compartment. The goat anti-LEF-1 (sc-8591) and anti-TCF-4 (sc-8631) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). (c) Phosphorylated and total p53 distribution and expression in nucleus and cytoplasm. The mouse monoclonal anti-p53 IC12 (no. 2524) and the rabbit polyclonal anti-Phospho-p53 (ser15) (no. 9284) antibodies were purchased from Cell Signaling Technologies (Boston, MA, USA). (d) Phosphorylated-RB expression in total cell lysate. The rabbit anti- phospho-RB (ser780) (no. 9307) antibody was purchased from Cell Signaling Technologies (Boston, MA, USA). To quantify the protein bands, we scanned the blots and performed a densitometry analysis, using the software NIH ImageJ v. 6.4 (freeware, NIH, MD, USA). Where the relative amount of protein is indicated as generic “ratio” instead of ratio to a specific housekeeping protein, it is because the protein quantization referred to the different cell compartment housekeepers indicated at the bottom of each blot. The figure shows a typical experiment of at least three performed under the same conditions.

Article Snippet: The mouse monoclonal anti-p53 IC12 (no. 2524) and the rabbit polyclonal anti-Phospho-p53 (ser15) (no. 9284) antibodies were purchased from Cell Signaling Technologies (Boston, MA, USA). (d) Phosphorylated-RB expression in total cell lysate.

Techniques: Expressing, Western Blot, Software

Journal: International journal of nephrology

Article Title: Constitutional Nephrin Deficiency in Conditionally Immortalized Human Podocytes Induced Epithelial-Mesenchymal Transition, Supported by β-Catenin/NF-kappa B Activation: A Consequence of Cell Junction Impairment?

doi: 10.1155/2013/457490

Figure Lengend Snippet: Figure 7: Effects of Bay-117082 on the distribution and expression of p53 and its phosphorylated form, phosphorylated pRB, 𝛽-catenin, a- SMA, vimentin, and p65 in wild type and nephrin-mutated podocytes. Where indicated, the cells were treated with Bay-117082 (Calbiochem, USA) at 0.5 mM for 4 days (in the figure indicated as Bay-11). Afterwards, they were lysated, fractionated, and processed for western blot as described in Section 2. The antibodies used have been described in the previous figures. To quantify the protein bands, we scanned the blots and performed a densitometry analysis, using the software NIH ImageJ v. 6.4 (freeware, NIH, MD, USA). The figure shows a typical experiment of at least three performed under the same conditions.

Article Snippet: The mouse monoclonal anti-p53 IC12 (no. 2524) and the rabbit polyclonal anti-Phospho-p53 (ser15) (no. 9284) antibodies were purchased from Cell Signaling Technologies (Boston, MA, USA). (d) Phosphorylated-RB expression in total cell lysate.

Techniques: Expressing, Western Blot, Software

Journal: International journal of nephrology

Article Title: Constitutional Nephrin Deficiency in Conditionally Immortalized Human Podocytes Induced Epithelial-Mesenchymal Transition, Supported by β-Catenin/NF-kappa B Activation: A Consequence of Cell Junction Impairment?

doi: 10.1155/2013/457490

Figure Lengend Snippet: Figure 8: Growth of wild type podocytes in Ca2+-free medium induced the same effects of nephrin ablation on 𝛽-catenin, p53, and phospho- pRB expressions and distribution. The cells were lysated, fractionated, and processed for western blot as described in Section 2. The antibodies used have been described in the previous figures. To quantify the protein bands, we scanned the blots and performed a densitometry analysis, using the software NIH ImageJ v. 6.4 (freeware, NIH, MD, USA). The figure shows a typical experiment of at least three performed under the same conditions.

Article Snippet: The mouse monoclonal anti-p53 IC12 (no. 2524) and the rabbit polyclonal anti-Phospho-p53 (ser15) (no. 9284) antibodies were purchased from Cell Signaling Technologies (Boston, MA, USA). (d) Phosphorylated-RB expression in total cell lysate.

Techniques: Western Blot, Software