p53 1c12  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Uncovering cancer dependencies in peptide-interacting protein pockets

doi: 10.64898/2026.03.13.711608

Figure Lengend Snippet: ( A ) Scheme showing how an overexpressed motif peptide from protein Y could outcompete the endogenous interaction between proteins X and Y and other interactions involving that pocket on protein X. ( B ) MDM2 SWIB domain binds to a degron motif in p53, leading to p53 degradation and maintenance of low p53 expression. ( C ) Superimposed structures of MDM2 SWIB domain in complex with the p53 degron peptide and Nutlin-3a. ( D ) Western blot images showing the dose-response of HCT116 cells to Nutlin-3a and p53 degron competitor peptides. ( E ) Flow cytometry plots showing the change in GFP-positive and mCherry-positive cell fractions in a pairwise competitive growth experiment in HCT116 cells. ( F ) Fusion with NLS and NES motifs localises the competitor peptides to different subcellular compartments, which impacts the anti-proliferative effect of the p53 peptide, as observed in a pairwise GFP/mCherry cell competitive growth experiment in HCT116. ( G ) Pairwise competitive growth experiments showing the impact of motif repeats on the potency of competitors based on NUMB MDM2 degron in HCT116. ( H ) Experimental scheme of proteome-wide competitor peptide screening to identify peptide-binding pocket inhibition vulnerabilities. ( I ) The distribution of peptide abundance changes in 9 cancer cell lines. The error bars show 5-95% percentile. ( J ) Heatmap showing dropout scores (log2 fold change) of the peptides identified as anti-proliferative in at least 7 cell lines.

Article Snippet: The following primary antibodies were used: ab13970 chicken polyclonal to GFP at 1:5000, p21 (12D1) Rabbit mAb (#2947, Cell Signaling Technologies) at 1:1000, p53 (1C12) Mouse mAb (#2524, Cell Signaling Technology) at 1:500, Hcfc1 Antibody (Amino-terminal Antigen) (#69690, Cell Signaling Technology) at 1:1000, TLE1 (F-4) (sc-137098, Santa Cruz Biotechnologies) at 1:500, TLE2 (D-10) (sc-374226, Santa Cruz Biotechnologies) at 1:500, TLE3 (D-10) (sc-514798, Santa Cruz Biotechnologies) at 1:500, TLE4 (E-10) (sc-365406, Santa Cruz Biotechnologies) at 1:500, TLE1/2/3/4 (#4681, Cell Signaling Technologies) at 1:500, ATF4 (PA5-27576, Thermo Fisher Scientific) at 1:2000, DDIT3 (R-20) (sc-793, Santa Cruz Biotechnologies) at 1:500, Vinculin (#13901, Cell Signaling Technology) at 1:1000, DHPS (A-10) (sc-365077, Santa Cruz Biotechnologies) at 1:500, ACOX3 (17360-1-AP, Proteintech) at 1:500, cyclin D1 (sc-20044, Santa Cruz Biotechnologies) at 1:500, cyclin D2 (D52F9) (#3741, Cell Signaling Technology) at 1:1000, cyclin D3 (DCS22) (#2936, Cell Signaling Technology) at 1:1000, eIF5A (D8L8Q) (#20765, Cell Signaling Technology) at 1:1000, hypusine (ABS1064-I, EMD Millipore) at 1:1000.

Techniques: Expressing, Western Blot, Flow Cytometry, Binding Assay, Inhibition

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a,b , Heatmap displaying relative abundance of all metabolites exhibiting significant ( P < 0.05) change when comparing KP sh cells cultured with or without dox for 6 days ( a ) or KP flox RIK cells expressing dox-inducible p53 compared with vector expressing cells cultured with dox for 2 days ( b ). Data can be found in Supplementary Table . c , Western blot of KP sh cells edited by the indicated sgRNA and cultured with or without dox for 6 days. d,e , Western blot of HCT116 cells ( d ) or wild-type p53 (p53 +/+ ) and p53 null (p53 −/− ) MEFs ( e ) cultured with DMSO (vehicle) or 5 μM Nutlin-3a (Nutlin) for 48 h. f , Senescence-associated β-galactosidase (SA-β GAL) staining in HCT116 cells cultured with DMSO (Control) or 5 μM Nutlin-3a (Nutlin) for 96 h. Scale bars as indicated. g , Steady-state levels of PEtn in KP sh cells cultured with dox and DMSO (vehicle) or 3 μM Etoposide (Etopo) for 96 h. h,i , Percentage of DAPI positive ( h ) or western blot ( i ) of KP sh cells cultured with or without dox for 6 days and Navitoclax (300 nM, 600 nM) for final 24 h. All experiments were repeated twice with similar results. Statistical significance was assessed by unpaired, two-tailed Student’s t- test ( a, b, g ). Data are mean ± SD, n = 3 independent replicates. Source numerical data and unprocessed blots are available in source data.

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: Cell Culture, Expressing, Plasmid Preparation, Western Blot, Staining, Control, Two Tailed Test

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , Schematic (top) and western blot (bottom) of KP sh cells cultured with or without dox for 6 days. b , Volcano plot showing log 2 fold change in metabolite abundance in KP sh cells with endogenous p53 activation (−Dox) compared with cells with silenced p53 (+Dox). PEtn and αKG are highlighted. c , Schematic (top) and western blot (bottom) of KP flox RIK cells expressing dox-inducible vectors cultured with or without dox for 2 days. Vec, empty vector. d , Volcano plot showing log 2 fold change in metabolite abundance in KP flox RIK cells described in c . e , PEtn levels in KP sh cells edited with the indicated sgRNA and cultured with or without dox for 6 days ( P = 1.4 × 10 −8 ). f , g , Steady-state levels of PEtn in HCT116 cells ( f ) or wild-type p53 (p53 +/+ ) and p53 null (p53 −/− ) MEFs ( g ) cultured with DMSO (vehicle) or 5 μM Nutlin-3a (Nutlin) for 48 h ( P values 0.0002 ( f ) and 1.2 × 10 −7 ( g )). h , PEtn levels in KP sh cells cultured with or without dox for 6 days and Navitoclax (300 nM, 600 nM) for final 24 h. All experiments were repeated twice with similar results. Statistical significance was assessed by unpaired, two-tailed Student’s t- test ( b , d and f ) or two-way ANOVA with Šidák’s multiple-comparisons post-test ( e , g and h ). Data are mean ± s.d., n = 3 independent replicates. The dotted line indicates a significance threshold of P < 0.05.

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: Western Blot, Cell Culture, Activation Assay, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , Schematic depicting de novo synthesis of PE and PC via the Kennedy pathway. [U- 13 C]glucose provides carbons (purple) that contribute to fatty acids (FAs) or glycerol used in de novo lipid synthesis. DAG, diacylglycerol. b , Relative pool size of major lipid classes in KP sh cells cultured on or off dox for 6 days ( P values <0.0001 (PE), 0.0112 (PC) and 0.0037 (PS)). Lipid abundance is normalized to protein content . c , Volcano plot showing log 2 fold change in lipid abundance following endogenous p53 activation in KP sh cells cultured with or without dox for 6 days. Lipid species are colour coded by lipid class. SM, sphingomyelin; Cer, ceramide. d , Heatmap depicting total fractional labelling of PE (top) or PC (bottom) species in KP sh cells cultured with or without dox for 6 days and [U- 13 C]glucose for 24 h. e , f , Average fraction labelled (left), fraction containing M + 2 n isotopologues (middle) or fraction containing M + 3 + 2 n isotopologues (right) from tracing experiment shown in d for PE ( e ) and PC ( f ). g , Levels of newly synthesized PE species from experiments shown in d – f . All experiments were repeated twice with similar results. Statistical significance was assessed by unpaired, two-tailed Student’s t- test ( b , c and g ) or paired, two-tailed Student’s t -test ( e and f ). Data are mean ± s.d. ( b and g ) or median and quartiles ( e and f ), n = 3 independent replicates. The dotted line on the y axis indicates significance threshold of P < 0.05.

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: Cell Culture, Activation Assay, Synthesized, Two Tailed Test

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , Western blot of KP flox RIK cells engineered to express doxycycline (dox)-inducible vectors containing wild-type p53 or p53 with mutations (m) in the first or second transactivation domains (TAD) grown with dox for 2 days. b , Violin plot showing log fold change in expression of HALLMARK p53 pathway genes activated by wild type p53 (log fold change > 0; P < 0.05) in KP flox RIK cells cultured with dox for 2 days. Log fold change is expressed relative to cells expressing empty vector. Statistical significance was assessed by paired two-tailed t -test using R. Data was plotted with median (centre line), 25 th -75 th percentiles and whiskers extending to data points within 1.5x the interquartile range. c , Heatmap showing expression of autophagy and lysosome genes in KP sh cells grown on or off dox for ten days and KP flox RIK cells cultured on dox for two days. Genes with P < 0.05 in KP sh cells are ranked according to fold change. d , Sequencing tracks for the Atg7 locus, displaying p53 ChIP-seq (top) and RNA-seq (bottom) signals in doxorubicin-treated primary MEFs (ref. , green) or KP flox RIK cells expressing vector or wild-type p53 treated with dox for 2 days (blue). e , Heatmap depicting normalized gene enrichment score from Gene set enrichment analysis (GSEA) of datasets representing different P53 activation models. Gene lists are provided in Supplementary Table . f , Western blot (short and long exposure) of KP sh cells cultured with or without 50 nM bafilomycin A1 (BafA) for 24 h and exposed to dox withdrawal for indicated time. g , PEtn levels in KP sh cells cultured without dox for the indicated number of days. h , Western blot of clonal KP sh cells expressing indicated sgRNA and cultured with or without dox for 6 days. i,j , Western blot ( i ) or PEtn levels ( j ) of KP sh cells cultured with or without dox for 6 days. Cells were treated with 25 nM Trametinib (Tram) for the final 48 h and/or 20 μM chloroquine (CQ) for the final 24 h of culture as indicated. For panel ( i ), Vinculin, p53 and pERK1/2 were run on the same gel, whereas ERK1/2 was run on a separate gel processed in parallel. All experiments were repeated twice with similar results. Data are mean ± SD, n = 3 independent replicates. Statistical significance was assessed by 1-way ANOVA with Tukey’s two-sided multiple comparisons post-test ( b ), two-sided Wald test ( c ), GSEA ( e ), or Dunnett’s multiple comparison post-test ( g ). Source numerical data and unprocessed blots are available in source data.

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: Western Blot, Expressing, Cell Culture, Plasmid Preparation, Two Tailed Test, Sequencing, ChIP-sequencing, RNA Sequencing, Activation Assay, Comparison

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , PEtn levels in KP flox RIK cells engineered to express dox-inducible vectors containing wild-type p53 (WT) or p53 with mutations (m) in the first or second transactivation domains (TAD) and grown with or without dox for 2 days ( P values: 7.06 × 10 −7 (WT) and 9.45 × 10 −7 (TAD2m)). b , Venn diagram displaying the overlap of genes significantly activated (log 2 fold change >0; P < 0.05) in three sets of comparative analyses: WT+Dox versus Vector KP flox RIK+Dox cells, WT+Dox versus TAD1/2m KP flox RIK+Dox cells, and −Dox versus +Dox KP sh cells. c , Bubble plot of pathway overrepresentation for genes induced in all three conditions depicted in the Venn diagram in b . GOBP, Gene Ontology Biological Process; GOCC, Gene Ontology Cellular Component; KEGG, Kyoto Encyclopedia of Genes and Genomes. d , Scatter plot comparing changes in gene expression with changes in p53 binding in KP flox RIK cells expressing wild-type p53 versus vector control. The dotted line indicates the threshold of log 2 fold change >1, with a curvature setting of 1.5. e , PEtn levels in KP sh cells cultured with or without dox for 6 days and DMSO (vehicle), 20 μM CQ or 50 nM bafilomycin A1 (BafA) for the final 24 h ( P values, left to right: 8.23 × 10 −5 and 0.0024). f , PEtn levels in HCT116 cells treated with DMSO (vehicle) or 5 μM Nutlin-3a (Nutlin) for 48 h with or without 20 μM CQ for the final 24 h ( P values, left to right: 0.0013 and 0.0009). g , PEtn levels in control and Atg7 -edited KP sh cells cultured with or without dox for 6 days ( P values, left to right: 5.36 × 10 −9 and 4.89 × 10 −10 ). Data are presented as mean ± s.d. of n = 3 independently treated wells, with individual data points shown ( a and e – g ). Statistical significance was assessed by two-way ANOVA with Šidák’s multiple-comparisons post-test ( a ), two-sided Wald test ( b ), Fisher’s exact test and adjusted for multiple testing using the Benjamini–Hochberg false discovery rate (FDR) ( c ), two-way ANOVA with Dunnett’s multiple-comparisons post-test ( e and g ) or one-way ANOVA with Tukey’s multiple-comparisons post-test ( f ).

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: Plasmid Preparation, Gene Expression, Binding Assay, Expressing, Control, Cell Culture

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , Heatmap of differentially expressed genes from RNA-Seq. Included genes exhibit an average FPKM greater than 1 across all conditions and P < 0.05 when comparing Pcyt2 -knockout (KO) to Pcyt2 KO + Pcyt2 cDNA cells in the context of p53 activation. See Supplementary Table for gene information. b , Scatter plots displaying the significance of transcription factors enriched around the promoters of genes upregulated in Pcyt2 -knockout compared to Pcyt2 -knockout + Pcyt2 cDNA under p53 activation, along with their respective log 2 fold changes. c , Representative transmission electron microscopy images of Pcyt2 -knockout (KO) KP sh cells, Pcyt2 -KO cells complemented with guide-resistant Pcyt2 cDNA, and parental controls, cultured on or off dox for 6 days. Scale bars as indicated for each column. d,e , Overall cell viability ( d ) and relative proliferation (e ) of Pcyt2 KO cells compared to Pcyt2 KO + Pcyt2 cDNA cells cultured on or off dox for 6 days. f , Senescence-associated β-galactosidase (SA-βGal) staining in KP sh cells with indicated genetic manipulations (left: representative images; right: quantification) cultured on or off dox for 6 days. Scale bars as indicated. Data are presented as mean ± S.D. of n = 3 independently treated wells with individual data points shown. Statistical significance was assessed by two-sided Wald test ( a ), unpaired, two-tailed Student’s t- test ( e ), two-way ANOVA with Tukey’s multiple comparison post-test ( d ) or Sidak’s multiple comparisons post-test ( f ). Source numerical data are available in source data.

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: RNA Sequencing, Knock-Out, Activation Assay, Transmission Assay, Electron Microscopy, Cell Culture, Staining, Two Tailed Test, Comparison

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , PEtn levels in parental KP sh and clonal KP sh cells expressing sgRNA and complemented with guide-resistant Pcyt2 cDNA as indicated cultured on or off dox for 6 days ( P values, left to right: 0.0183, 0.0341, 0.0068 and 0.0104). b , c , PCA of RNA-seq ( b ) and bubble plot of pathway overrepresentation for genes that are upregulated in KP sh Pcyt2 -KO cells relative to cells complemented with sgRNA-resistant Pcyt2 cDNA following 6 days of dox withdrawal to activate p53 ( c ). d , Representative transmission electron microscope images from Pcyt2 -KO or Pcyt2 -KO complemented with guide-resistant Pcyt2 cDNA KP sh cells cultured on or off dox for 6 days. Scale bars as indicated. e , Population doublings of Pcyt2 -KO or Pcyt2 -KO complemented with guide-resistant Pcyt2 cDNA KP sh cells cultured on or off dox for indicated times. f , g , Tumour growth curve ( f ) or endpoint tumour volumes ( g ) for Pcyt2 -KO and Pcyt2 -KO complemented with guide-resistant Pcyt2 cDNA KP sh cell line xenograft maintained on dox chow ( sgPcyt2 , n = 8; sgPcyt2 + cDNA, n = 10) or withdrawn from dox chow ( sgPcyt2 , n = 12; sgPcyt2 + cDNA, n = 12) for 3 weeks. Otherwise, data represent n = 3 independently treated wells. Data are presented as mean ± s.d. ( a , e and g ) or s.e.m. ( f ), and statistical significance was assessed by two-sided Holm–Šídák method for multiple unpaired t- tests ( a ), Fisher’s exact test and adjusted for multiple testing using the Benjamini–Hochberg false discovery rate ( c ) or unpaired, two-tailed Student’s t- test ( g ). * P < 0.05, ** P < 0.01.

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: Expressing, Cell Culture, RNA Sequencing, Transmission Assay, Microscopy, Two Tailed Test

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , Scatter plot comparing CRISPR gene scores KP sh cells cultured with or without dox for 10 days. Genes more essential following p53 activation ([gene score −dox] – [gene score +dox] < −1) are highlighted in orange. Pearson correlation (two-sided) r = 0.66, P < 1 × 10 −15 . All data are presented in Supplementary Table . b , Bubble plot of pathway enrichment for differentially essential genes highlighted in orange in a . GOBP, Gene Ontology Biological Process; GOCC, Gene Ontology Cellular Component. c , STRING network of differentially essential genes highlighted in orange in a . d , Population doublings of KP sh cells treated with 5 μM CQ and cultured with or without dox for the indicated time. e , f , Population doublings of KP sh cells edited with sgRNA targeting Cers2 ( e ) or Hnf4a sgRNA ( f ) or a control region (Chr8), cultured with or without dox for the indicated times. Cells were pretreated with or without dox for 2 days before seeding ( P values— Cers2 : sg1, 1.64 × 10 −5 ; sg2, 2.43 × 10 −8 ; sg3, 3.41 × 10 −8 ; Hnf4a : sg1, 8.73 × 10 −6 ; sg2, 2.07 × 10 −5 ; sg3, 1.86 × 10 −4 ). Data are presented as mean ± s.d. of n = 3 independently treated wells, with individual data points shown. Statistical significance was assessed by Fisher’s exact test and adjusted for multiple testing using the Benjamini–Hochberg false discovery rate ( b ) or two-way ANOVA with Dunnett’s multiple-comparison post-test relative to Chr8 ( e and f ).

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: CRISPR, Cell Culture, Activation Assay, Control, Comparison

Journal: Nature Cell Biology

Article Title: p53 increases phospholipid headgroup scavenging in senescence

doi: 10.1038/s41556-025-01853-0

Figure Lengend Snippet: a , Schematic depicting the metabolism-focused CRISPR genetic screens in KP sh cells. Cells were sequenced at day 0 (prior to dox withdrawal) and then after 10 days of additional growth with or without dox. b , Scatter plot showing normalized counts for each gene at day 0 (x-axis) and log 2 foldchange in normalized counts between day 10 and day 0 (y axis). Left: p53 off, right: p53 on. Genes highlighted in orange are genes differentially essential in p53-on cells, as shown in Fig. . c , Scatter plot comparing changes in gene expression induced by p53 activation (x-axis, log 2 fold change in expression KP sh cells cultured -dox compared to cells cultured +dox) with changes in gene scores following p53 activation (y-axis, gene scores determined by comparing sgRNA abundance of cells following 10 days culture with or without dox). Pearson correlation (two-sided) r = −0.10, P < 5.2 ×10 −8 . Genes both induced by p53 and more essential following p53 activation (log 2 fold change > 0.4; screen enrichment difference < −0.2) are highlighted in orange. Data are available in Supplementary Table . d , Bubble plot of pathway enrichment for genes highlighted in ( c ). e , Volcano plot showing pathways from gene set enrichment analysis (GSEA) analysis of genes co-expressed with HNF4A in pancreatic cancer cell lines in DepMap. Each dot present one pathway. f , GSEA plot showing lipid metabolism related pathways enriched among genes exhibiting similar expression patterns as HNF4A . g,h , Normalized counts individual sgRNA targeting Cers2 ( g ) and Hnf4a ( h ) in indicated conditions from the screen (P value: g = 0.0469, h = 0.0078). i , Scatter plot of DepMap gene effect versus log2 fold change in gene score (-dox/+dox at day 10) in KP sh cells. Each dot represents a gene; Cers2 and Hnf4a are highlighted in cyan and blue. j,k , Western blot of KP sh cells expressing sgRNA targeting Cers2 ( j ) and Hnf4a ( k ). Statistical significance was assessed by Fisher’s Exact test and adjusted for multiple testing using the Benjamini-Hochberg false discovery rate ( d ), GSEA ( e ), or Wilcoxon matched-pairs two-tails signed rank test ( g, h ). Source numerical data and unprocessed blots are available in source data.

Article Snippet: Subsequently, 5.5 μg of p53 (1C12) antibody (Cell Signaling Technology) and 55 μl of Dynabeads Protein G (Thermo Fisher Scientific) were used for each ChIP.

Techniques: CRISPR, Gene Expression, Activation Assay, Expressing, Cell Culture, Western Blot

Journal: Cell Death & Disease

Article Title: Mice carrying nonsense mutant p53 develop frequent multicentric or metastatic tumors

doi: 10.1038/s41419-025-08290-9

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 with numerous mitoses (asterisks), and dispersed, moderate numbers of tingible body macrophages (arrows) efface the normal thymic architecture. Scale bars = 50 µm. B Flow cytometry to verify T-cell origin of X405 cells derived from a Trp53 R210X/R210X thymic lymphoma by staining with antibodies for CD3 (left panel), and CD4 and CD8 (right panel). C Immunofluorescence staining of X405 T-lymphoma cells, either untreated (0 µM G418, left panels) or treated with 100 µM G418 (right panels) for 72 h. p53 was detected using N1 (N-terminal epitope) and 280aa C-term (C-terminal epitope) antibodies. Bottom 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 = 100 µm. D Western blot analysis showing dose-dependent induction of full-length p53 in X405 T-lymphoma cells following 72 h treatment with G418 at indicated concentrations. 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 and p53 target gene mRNA levels in the three Trp53 R210X/R210X T-lymphoma cell lines X405, X491 and X547 after 72 h treatment with G418 (upper panel). Two independently established Trp53 R172H/R172H T-lymphoma cell lines were used as negative control (lower panel). Gene expression values were normalized to Gapdh expression and compared to the untreated (0 µM G418) negative control for each gene. Each dot represents the mean from three independent experiments per cell line. F WST-1 assay to determine proliferation of T-lymphoma cells from Trp53 R210X/R210X ( n = 3 cell lines) and Trp53 R172H/R172H ( n = 2 cell lines) mice upon 72 h treatment with indicated concentrations of G418. Each dot represents the mean from eight independent experiments per cell line. G Flow cytometry of Annexin V staining to assess apoptotic cell death in Trp53 R210X/R210X ( n = 3 cell lines) and Trp53 R172H/R172H ( n = 2 cell lines) T-lymphoma cells following 72 h treatment with G418. All quantifications from three independent experiments per cell line are shown. Statistical analysis was performed by repeated measures two-way ANOVA followed by Dunnett’s multiple comparisons test (E) , and by two-way Mixed-effects analysis (F , G ), respectively. All individual cell line values from all experiments were used for statistical analysis but mean ± SEM are indicated in ( E , F ). Adjusted P-values: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Detection of p53 was performed using primary antibody 1C12 anti-p53 (1:1000, #2524, Cell Signaling Technology) and an HRP-conjugated Rabbit-anti-Mouse IgG secondary antibody (1:5000, #61-6520, Invitrogen, USA).

Techniques: Staining, Flow Cytometry, Derivative Assay, Immunofluorescence, Mutagenesis, Western Blot, Control, Quantitative RT-PCR, Negative Control, Gene Expression, Expressing, WST-1 Assay