Journal: bioRxiv

Article Title: Mice carrying nonsense mutant p53 develop spontaneous tumors with frequent metastases

doi: 10.1101/2025.03.12.642796

Figure Lengend Snippet: (A) Representative histomicrographs of the thymus from the Trp53 R210X/R210X mouse X405 stained with HE (left) and CD3 antibody (IHC, right). Mats of CD3-positive, i.e. T-cell origin, pleomorphic neoplastic lymphocytes and dispersed, moderate numbers of tangible body macrophages (left panel, arrows) efface the normal thymic architecture. Scale bars = 50µm. (B) Representative flow cytometry to verify T-cell origin of the X405 Trp53 R210X/R210X T-lymphoma cell line established from a thymic lymphoma by staining with antibodies for CD3 (left panel), and CD4 and CD8 (right panel). (C) Immunofluorescence staining of X405 Trp53 R210X/R210X T-lymphoma cells, either untreated (NT, upper panels) or treated with 100µM G418 (lower panels) for 72 hours. p53 was detected using N1 (N-terminal epitope) and 280aa C-term (C-terminal epitope) antibodies. Rightmost panels show merged DAPI and 280aa C-term p53 staining, highlighting translational readthrough of the Trp53 -R210X nonsense mutation and production of full-length p53 protein upon treatment with G418. Scale bars = 100µm. (D) Western blot analysis showing dose-dependent induction of full-length p53 in X405 Trp53 R210X/R210X T-lymphoma cells following 72h treatment with G418. p53 was visualized using the anti-p53 antibody 1C12 that recognizes an N-terminal epitope and therefore detects both full-length and C-terminally truncated p53. Gapdh was used as a loading control. (E) qRT-PCR analysis showing dose-dependent induction of p53 mRNA levels in X405 Trp53 R210X/R210X T-lymphoma cells after 72h treatment with G418. mRNA levels of p53 target genes p21 , Puma and Zmat3 were significantly upregulated upon treatment with 200µM G418. Gene expression values were normalized to Gapdh expression and compared to the untreated (NT) negative control. (F) Representative flow cytometry for assessment of cell death by Annexin V staining of X405 Trp53 R210X/R210X T-lymphoma cells (left) and quantification of 3 flow cytometry replicates (right), showing significant increase in cell death following treatment with 200µM G418 for 72h. In E-F : statistical analysis was performed using one-way ANOVA followed by Dunnett’s (E) or Tukey’s (F) multiple comparisons tests. Values represent the mean ± SEM. Adjusted P-values: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Primary antibodies rabbit polyclonal p53 N-terminal antibody [N1] (GTX100629, Genetex, USA) and goat polyclonal Anti-TP53 (280aa C-Term) (ASJ-VN4O74-150, Nordic Biosite, Sweden) were diluted together at 1:100 in PBS, incubation 4°C ON.

Techniques: Staining, Flow Cytometry, Immunofluorescence, Mutagenesis, Western Blot, Control, Quantitative RT-PCR, Gene Expression, Expressing, Negative Control