Journal: Oncogene

Article Title: Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

doi: 10.1038/s41388-021-01847-w

Figure Lengend Snippet: A – C Gene expression profiling by RNA-seq of SW480 cells after transduction with either shTGM2-1 or shSCRMBL. A Unsupervised hierarchical clustering of the top 1000 differentially expressed genes (DEGs) upon TGM2 knockdown across the four biological replicates. B MA plot relating p values for all differentially expressed genes between shTGM2-1 and shSCRMBL from four biological replicates. Red dots indicate significantly regulated genes (adjusted P < 0.05). List of regulated genes is presented in Supplementary Table S . C Scatter plot of gene set enrichment analysis of DEGs relating the Q-value for Hallmark gene-set signatures. The top 16 enriched pathways are shown ( P < 0.05, Fold change ≥2). The color and size of each dot represent the Rich factor and the number of DEGs mapped to the indicated pathway, respectively. D Proteome analysis of regulated proteins involved in apoptosis upon shRNA-mediated TGM2 knockdown. Representative blot of Proteome Profiler Array™-Human Apoptosis Array analysis of SW480 cells. The regulation of protein expression of phosphorylated p53 variants is shown. E – H Quantification of p53 and phosphorylated p53 (S15, S46, and S392) upon TGM2 knockdown in SW480 ( E , G ) and HCT-116 ( F , H ) cells via Simple Western technology ( n = 3; Mann–Whitney U test).

Article Snippet: The p53 knockout colon cancer cell line HCT-116 (p53 −/− ) was obtained from Accegen Biotechnology (Köln, Germany).

Techniques: Gene Expression, RNA Sequencing, Transduction, Knockdown, shRNA, Expressing, Simple Western, MANN-WHITNEY

Journal: Oncogene

Article Title: Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

doi: 10.1038/s41388-021-01847-w

Figure Lengend Snippet: A Representative images of proximity ligation assay (PLA) of TGM2 and p53 in SW480 cells. Cells incubated only with TGM2 antibody served as negative control (I). Protein–protein interaction of TGM2 and p53(S15) was visualized using hybridization probes labeled with Texas Red (II). Nuclei were stained with DAPI (blue). B Quantification of TGM2-p53 interaction and associated technical controls (Ctrl). Technical controls demonstrate the specificity of PLA signals. Each dot represents one cell. Mean value of PLA dots per cell is shown by the black line. C Representative images of proximity ligation assay of TGM2 and p53 in patient-derived normal epithelial cells (I) and corresponding colon cancer cells (II). D Quantification of TGM2-p53 interaction in primary patient material. (Significance was calculated using Kruskal–Wallis test). E Co-immunoprecipitation (Co-IP) of endogenous TGM2 and p53 or phosphorylated p53(S15) in SW480, HCT-116 p53 wildtype cells (wt) and HCT-116 p53 knockout cells (−/−). F Super-resolved image of a HCT-116 cell immunostained for TGM2 (red) and p53(S15) (cyan). A zoom-in of the highlighted region is shown on the right. White regions indicate overlapping signal of TGM2 and p53(S15) (yellow arrowheads). Scale bars represent 5 µm and 1 µm, respectively.

Article Snippet: The p53 knockout colon cancer cell line HCT-116 (p53 −/− ) was obtained from Accegen Biotechnology (Köln, Germany).

Techniques: Proximity Ligation Assay, Incubation, Negative Control, Hybridization, Labeling, Staining, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Knock-Out

Journal: Oncogene

Article Title: Transglutaminase 2 promotes tumorigenicity of colon cancer cells by inactivation of the tumor suppressor p53

doi: 10.1038/s41388-021-01847-w

Figure Lengend Snippet: A – C HCT-116 p53 wildtype cells (wt) and HCT-116 p53 knockout cells (−/−) were transduced with either shTGM2-1, shTGM2-2, or shSCRMBL. Time-lapse imaging and proliferation assay were performed to determine a rescue from cell death upon TGM2 knockdown. A Fold change of cell number of HCT-116 p53 wt and HCT-116 p53 −/− cells upon TGM2 knockdown in comparison to shSCRMBL control determined at day three after transduction. Data are presented as mean ± SD of three independent experiments (** P < 0.01, Mann–Whitney U test). B Single cell tracking of HCT-116 p53 wt and HCT-116 p53 − /− cells after TGM2 knockdown with shTGM2-1 and (C) shTGM2-2. Cumulative cell death events are shown over time (*** P < 0.001, Log-rank test). D Direct visualization of p53 activation upon TGM2 knockdown by time-lapse video-microscopy. Sequence of phase contrast images, tdTOMATO fluorescence of shTGM2-1 and p53-driven destabilized GFP reporter , depicting the same field of view over the time course of 30 hours as indicated in the corresponding panels in I–VIII. The yellow circles designate tracked cells over time. (I–VIII) show corresponding sequence of fluorescence images taken at the same time points as the phase contrast images. (I) Shown are two representative HCT-116 cells. (II and III) 6-8 hours after lentiviral transduction of shTGM2-1 both HCT-116 cells express the red fluorescent tdTOMATO reporter, indicating a knockdown of TGM2. (IV-VI) Another 4–10 hours later both cells express the green fluorescent (GFP) p53 reporter, indicating the induction of p53 activity. (VII and VIII) About 24 hours after transduction both HCT-116 cells subsequently undergo apoptosis (white arrows). Movie S shows all assembled images (3 min temporal resolution) of the same sequence.

Article Snippet: The p53 knockout colon cancer cell line HCT-116 (p53 −/− ) was obtained from Accegen Biotechnology (Köln, Germany).

Techniques: Knock-Out, Transduction, Imaging, Proliferation Assay, Knockdown, Comparison, Control, MANN-WHITNEY, Single Cell Tracking, Activation Assay, Microscopy, Sequencing, Fluorescence, Activity Assay

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: puma −/− ; noxa −/− ; p21 −/− zebrafish are not predisposed to spontaneous tumors (A) Kaplan-Meier tumor-free survival of p53 −/− (blue curve, N = 96, T50 = 261 days) zebrafish compared with puma −/− ; noxa −/− ; p21 −/− (called pnp −/− , N = 43, green) and wildtype allele ( N = 96, orange). (B) Kaplan-Meier tumor-free survival of BRAF V600E ; p53 −/− (blue curve, N = 44, T50 = 271 days) zebrafish compared with BRAF V600E ; puma −/− ; noxa −/− ; p21 −/− (called BRAF V600E ; pnp −/− , N = 42, green), BRAF V600E ; p21 −/− ( N = 52, green), and wildtype allele ( N = 44, orange). Long-rank statistic test was done. ∗∗∗∗, p -value between p53 −/− and pnp −/− < 0.0001 and p -value between p53 −/− and p53 +/+ < 0.0001. ∗∗∗∗, p -value between BRAF V600E ; p53 −/− and BRAF V600E ; pnp −/− < 0.0001, p -value between p -value between BRAF V600E ; p53 −/− and BRAF V600E ; p21 −/− < 0.0001, and BRAF V600E ; p53 −/− and BRAF V600E ; p53 +/+ < 0.0001.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques:

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Loss of puma , noxa , and p21 provide resistance to p53-mediated induction of apoptosis and partially resistance to p53-mediated cell-cycle arrest (A) Experimental workflow showing how samples were harvested. 29-, 27- and 24-h post fertilization (hpf) wildtype, puma −/− ; noxa −/− , pnp −/− and p53 −/− zebrafish embryos were treated with 30 Gy IR-irradiation and fixed at 1-, 3-, 6-, 9- and 12-h post IR-treatment (hpi, 1hpi, 3hpi and 6hpi panels). (B) Representative images of anti-active Caspase-3 staining on 30-hpf zebrafish embryos for each group. Arrows in WT points out active apoptotic area in head region at 3 and 6 hpi. Scale bar: 500μM. (C) Representative images of phospho-histone H3 (pH3)-stained 30-hpf (1 and 3 hpi) or 36-hpf (12 hpi) zebrafish embryos for each group. Experimental design showing in A and A. Scale bar: 500μM. (D) Quantification of pH3 positive cells in treated and untreated WT, pnp −/− and p53 −/− embryos for each group. Each dot represents an individual. The average number of pH3+ cells (Mean) were indicated in each group. Bars represent mean ± SEM. ∗, p < 0.05. ∗∗∗, p < 0.001.∗∗∗∗, p < 0.0001.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Irradiation, Staining

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Loss of p21 partially rescues p53 -dependent mdm2 -null induced cell-cycle arrest (A) The conceptional diagram of mdm2 -null induced embryonic lethality. Loss of mdm2 elevates p53 protein levels to induce downstream targets and effector functions to render the lethality. (B) Representative gross images of 24-hpf mdm2 +/+ , mdm2 −/− ; puma −/− ; noxa −/− ; p21 −/− ( mpnp −/− ) and mdm2 −/− ; p53 −/− embryos. Scale bar: 500μM. (C) pH3-stained mdm2 +/+ , mdm2 −/− ; mpnp −/− embryos at 12-, 16-, 20- and 24-hpf. Scale bar: 200μM. (D) Quantification of pH3 positive cells at 12 hpf (Top panel) and at 24 hpf (bottom panel). Each dot represents an individual. Bars represent mean ± SEM. ∗∗∗, p < 0.001.∗∗∗∗, p < 0.0001. Not statistical significance between mdm2 −/− and mpnp −/− at 24 hpf.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Staining

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Defining IR induced zebrafish early responsive p53 -upregulated genes (A and B) Volcano plots showing 30-hpf zebrafish pnp −/− embryos with the treated versus untreated at 1 (A) and 3 hpi (B). The cutoff was set as fold change ≥2 or ≤ −2 and p value <0.05. Upregulated DEGs were color-labeled with magenta and the downregulated were labeled with blue. The gene symbol of some TOP DEGs was indicated on the plot. The -log 10 ( p -value) of phlda3 , foxo3b and mdm2 treated versus untreated at 3 hpi is above 300. Their log 2 (Fold change) values were pointed out (top right square). (C) Schematic of the method used to create Venn diagrams for p53 -upregulated DEGs in pnp −/− at 1 and 3 hpi (left panel). Venn graphs for the DEGs (right panel). The cut-off is fold change ≥1.5 and q < 0.05. (D) Venn graph showing 264 p53 -induced genes in pnp −/− between 1 and 3 hpi. (E) Representative plots showing well-established p53 targets, including gadd45aa , mdm2 and ccng1 , in pnp −/− but not p53 −/− datasets in response to IR treatment.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Labeling

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Defining conserved p53-upregulated genes in zebrafish and mouse (A) Venn graphs representing p53 -upregulated DEGs in mouse p53 +/+ at 1 and 3 hpi. The cut-off is fold change ≥1.5 and q < 0.05. (B) Venn graph showing 1,617 p53 -induced genes in mouse p53 +/+ between 1 and 3 hpi. (C) The diagram showing the analysis on mouse orthologs of p53-upregulated DEGs in pnp −/− zebrafish embryos at 1 and 3 hpi. For 264 p53-upregulated DEGs defined in zebrafish at 1 or 3 hpi, 247 of them are with mouse orthologs. Among them, 226 genes have one ortholog, and 21 of them are with multiple orthologs. 12 did not define orthologs in mouse. Five of them are non-coding genes. And 247 zebrafish p53-upregulated DEGs are corresponding to 323 mouse orthologs and 1,804 mouse paralogs. Among them, 232 genes are upregulated in mouse WT but not in p53 −/− treated versus untreated. Finally, defining 137 zebrafish p53 -induced DEGs are also conserved upregulated by p53 in mouse.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques:

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: Comparing 137 conserved p53 dependent IR induced genes with DEGs in mpnp −/− datasets (A) Venn graph displaying the overlapping genes between 137 conserved UP DEGs in both zebrafish and mouse with IR-irradiation and 2,582 upregulated (UP) DEGs in mpnp −/− versus sibling controls at 18 hpf. Experimental timeline showing the timepoint that distinguish mpnp −/− embryos from sibling controls and how to harvest RNA samples at the time (Top panel). (B) Heatmap showing transcriptional changes for 2,582 DEGs over time in early mpnp −/− datasets (8, 10, 12, 14, and 16 hpf). Each timepoint was measured in duplicate, and the averages of the duplicates were used for each group. Z-scores, calculated from TPM values calculated across all samples, were used to standardize gene expression levels before clustering. The experimental workflow for sample collection is shown in the top panel. Note that mutant samples at each timepoint were diluted 4-fold by their sibling controls (¼ mpnp −/− and ¾ sibling controls). Genes with similar expression patterns over time were grouped into eight clusters, with their trends and cluster sizes shown in the right panel. (C) Line graphs showing the kinetics of 8 out of 24 GOIs in early mpnp datasets, with the dashed line indicating a fold change of 2. Expected counts calculated with RSEM were used for creating line graphs. Line graphs for the remaining 16 GOIs are shown in B.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Irradiation, Gene Expression, Mutagenesis, Expressing

Journal: iScience

Article Title: p21 , ccng1 , foxo3b , and fbxw7 contribute to p53 -dependent cell cycle arrest

doi: 10.1016/j.isci.2025.112558

Figure Lengend Snippet: fbxw7 , foxo3b and ccng1 G0 crispants mitigate p53 -mediated cell-cycle arrest (A) Representative images showing pH3-stained un-injected (control, un-inj) and injected mpnp −/− embryos at 21 hpf. Scale bar: 250μM. (B) Quantification of pH3 positive cells in injected mpnp −/− embryos for 24 GOIs. un-inj (negative control) and the p53 guides-injected ( p53 , positive control). (C) Representative images representing pH3-stained, IR-irradiation treated or untreated, four-guide injected pnp −/− embryos at 30 hpf. Scale bar: 500μM. (D) Quantification of pH3 positive cells in injected pnp −/− embryos for fbxw7 , foxo3b and ccng1 . p21 -inj (negative control) and p53 -inj (positive control). Each dot represents an individual. Bars represent mean ± SEM. ∗, p < 0.05. ∗∗, p < 0.01.∗∗∗, p < 0.001.∗∗∗∗, p < 0.0001.

Article Snippet: The p53 knockout (KO) allele was obtained from Jackson Labs (Strain #002101) and maintained on a C57BL6/J genetic background (Strain #000664), which were also purchased from Jackson Labs. Mice had ad libitum access to food and water under 12:12 light/dark cycle at 21-22°C.

Techniques: Staining, Injection, Control, Negative Control, Positive Control, Irradiation

Journal: Cancer Science

Article Title: Regulation of the innate immune response and gut microbiome by p53

doi: 10.1111/cas.15991

Figure Lengend Snippet: p53 downstream targets are enriched for immune system genes. (A) Gene Ontology (GO) enrichment analysis for p53‐induced genes with ≥5‐fold‐change (FC) upregulation in one tissue. p ‐value is Benjamini‐corrected Fisher's exact test. (B) FC of genes ( n = 57) annotated in the innate immunity GO term in 24 mouse tissues. FC is mean gene expression from the p53 +/+ WBI over the maximum of the other three groups ( p53 +/+ , p53 −/− , and p53 −/− WBI; n = 3 each).

Article Snippet: p53‐knockout mice ( p53 −/− ; BRC no.: RBRC00107) with C57BL/6 background were provided by RIKEN BioResource Center.

Techniques: Gene Expression

Journal: Cancer Science

Article Title: Regulation of the innate immune response and gut microbiome by p53

doi: 10.1111/cas.15991

Figure Lengend Snippet: p53 −/− mice exhibited an altered gut microbiome. (A) Overall relative abundance of taxa at genus level of the gut microbiome between p53 +/+ and p53 −/− littermates at 48 h following 20 Gy WBI. (B) Class comparisons of the gut microbiome between p53 +/+ and p53 −/− littermates at 48 h following 20 Gy WBI, performed using linear discriminant analysis effect size (LEfSe). Bacterial taxa at all taxonomic levels, differentially abundant between p53 +/+ WBI and p53 −/− WBI, are shown. (C) Histogram of the linear discriminant analysis (LDA) score shows the breakdown of taxa enriched in the p53 +/+ WBI and p53 −/− WBI gut microbiomes at 48 h following 20 Gy WBI, from LEfSe analysis. (D) p53 +/+ and p53 −/− are fecal samples of the same mice collected before 20 Gy WBI. (E) Short‐chain fatty acid (SCFA) levels in p53 +/+ WBI and p53 −/− WBI littermates at 48 h following 20 Gy WBI. p53 +/+ and p53 −/− are fecal samples collected from the same mice before WBI ( n = 4 each).

Article Snippet: p53‐knockout mice ( p53 −/− ; BRC no.: RBRC00107) with C57BL/6 background were provided by RIKEN BioResource Center.

Techniques:

Journal: Cancer Science

Article Title: Regulation of the innate immune response and gut microbiome by p53

doi: 10.1111/cas.15991

Figure Lengend Snippet: Gut microbiota depletion by antibiotics confers a protective effect on p53 −/− mice. (A) Kaplan–Meier curve shows the survival of p53 +/+ ( n = 14) and p53 −/− ( n = 11) mice following 20 Gy WBI. (B) Representative image of the gastrointestinal (GI) tract for p53 +/+ and p53 −/− mice 72 h after 20 Gy WBI. (C) Kaplan–Meier curve shows the survival of p53 −/− ( n = 10), p53 +/+ ( n = 12) and p53 −/− mice treated with antibiotics ( n = 11) and exposed to 20 Gy WBI. (D) Representative GI tract image of p53 −/− and p53 +/+ mice with antibiotic treatment and p53 −/− and p53 +/+ without antibiotic treatment at 72 h after 20 Gy WBI. The mouse jejunum was evaluated by H&E staining. Representative images are shown. Right, 100× magnification; left, 200× magnification of the boxed area on the left. H&E, hematoxylin and eosin. (A, C) p ‐values are from log‐rank test.

Article Snippet: p53‐knockout mice ( p53 −/− ; BRC no.: RBRC00107) with C57BL/6 background were provided by RIKEN BioResource Center.

Techniques: Staining

Journal: Cancer Science

Article Title: Regulation of the innate immune response and gut microbiome by p53

doi: 10.1111/cas.15991

Figure Lengend Snippet: Mbl2 and Lcn2 regulation by p53 in the digestive system of mice. (A) Fold change (FC) of genes annotated in the innate immunity Gene Ontology (GO) term on the tissue of the digestive system. Fold change is the mean gene expression from the p53 +/+ WBI group over the maximum of the other three groups ( p53 +/+ , p53 −/− , and p53 −/− WBI; n = 3 each). Transcriptome data of (B) Mbl2 and (C) Lcn2 mRNA expression in mouse tissues of the digestive system. RNA‐seq was performed on tissues from four groups of mice: p53 +/+ , p53 −/− , p53 +/+ WBI, and p53 −/− WBI ( n = 3). Ten Gy of X‐ray was used for WBI, and tissues were collected 24 h later. (D) Relative Mbl2 (left) and Lcn2 (right) mRNA levels in mouse livers and (E) relative Mbl2 mRNA level in mouse small intestine 24 h after 10 Gy WBI, assessed by RT‐qPCR. mRNA expression was normalized to Gapdh . (F) Western blot analysis of Mbl2 and Lcn2 protein in mouse liver, small intestine, and plasma. Samples were collected 24 h after 10 Gy WBI. β‐Actin was used as a loading control. Mouse plasma was loaded at 5 μL per well for each sample ( n = 3 each). (G) ELISA of Mbl2 (left; n = 3 each) and Lcn2 (right; p53 +/+ and p53 +/+ WBI, n = 5 each; p53 −/− , n = 6; p53 −/− WBI, n = 8) plasma levels at 24 h after 10 Gy WBI. (H) Representative image of p53 +/+ and p53 −/− mice liver 72 h after 20 Gy WBI, evaluated by H&E staining. 400× magnification. (I) Immunohistochemical analysis of CD45 in liver of p53 +/+ and p53 −/− mice 72 h after 20 Gy WBI. 400× magnification (J) Cxcl10 mRNA expression in mice liver from four groups of mice: p53 +/+ , p53 −/− , p53 +/+ WBI, and p53 −/− WBI ( n = 3 each). Ten Gy of X‐ray was used for WBI, and tissues were collected 24 h later. Error bars are the means ± SDs. SD, standard deviation. Error bars are the means ± SDs. Antibodies used in immunoblotting and Immunohistochemistry are listed in Table .

Article Snippet: p53‐knockout mice ( p53 −/− ; BRC no.: RBRC00107) with C57BL/6 background were provided by RIKEN BioResource Center.

Techniques: Gene Expression, Expressing, RNA Sequencing, Quantitative RT-PCR, Western Blot, Clinical Proteomics, Control, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Standard Deviation, Immunohistochemistry

Journal: Cancer Science

Article Title: Regulation of the innate immune response and gut microbiome by p53

doi: 10.1111/cas.15991

Figure Lengend Snippet: Binding of Mbl2 and Lcn2 by p53 in mice. (A, B) Location of the p53 binding sites (BSs; top) identified in the Mbl2 and Lcn2 gene regions. Nucleotides in the BSs modified for mutated BS constructs are indicated in red. Luciferase assay (bottom) performed in H1299 cells using the reporter vector pGL4.24, wild‐type BS construct, or mutated BS constructs. BS constructs were tested for p53 binding with wild‐type mouse p53 and mutant p53 (p53R172H) expression vectors. Empty vector pcDNA3.1 was used as a control. Relative luciferase activity indicates the luciferase activity normalized to the control vectors pcDNA3.1 and pGL4.24. The maroon bar denotes public p53 ChIP‐seq peaks obtained from the ReMap2022 database. The light blue line denotes the identified p53 BS scanned with JASPAR p53 transcription factor (TF)‐binding profiles.

Article Snippet: p53‐knockout mice ( p53 −/− ; BRC no.: RBRC00107) with C57BL/6 background were provided by RIKEN BioResource Center.

Techniques: Binding Assay, Modification, Construct, Luciferase, Plasmid Preparation, Mutagenesis, Expressing, Control, Activity Assay, ChIP-sequencing

Journal: Cancer Science

Article Title: Regulation of the innate immune response and gut microbiome by p53

doi: 10.1111/cas.15991

Figure Lengend Snippet: p53 binding sites identified in Mbl2 , MBL2 , Lcn2, and LCN2 genes.

Article Snippet: p53‐knockout mice ( p53 −/− ; BRC no.: RBRC00107) with C57BL/6 background were provided by RIKEN BioResource Center.

Techniques: Binding Assay, Amplification, Reporter Assay

Journal: Cancer Science

Article Title: Regulation of the innate immune response and gut microbiome by p53

doi: 10.1111/cas.15991

Figure Lengend Snippet: MBL2 and LCN2 regulation and binding by p53 in humans. (A) Mean relative MBL2 (left) and LCN2 (right) mRNA expression normalized to ACTB in HepG2 cells transfected with siRNA targeting enhanced green fluorescent protein (EGFP) or p53 ( n = 2). siRNA against EGFP (negative control) was used for comparison. (B) MBL2 and LCN2 mRNA expression in liver hepatocellular carcinoma tissue with p53‐wild‐type (p53‐Wt) and p53‐mutant (p53‐Mt) gene. Liver hepatocellular carcinoma data were obtained from TCGA database. Group comparisons were analyzed by two‐tailed Student's t‐ test; * p < 0.05. (C, D) p53 binding sites (BSs) locations (top) identified in the MBL2 and LCN2 gene regions. Nucleotides in the BSs modified for mutated BS constructs are indicated in red. Luciferase assay (bottom) was performed in H1299 cells using the reporter vector pGL4.24, wild‐type BS construct, or mutated BS constructs. BS constructs were tested for p53 binding with wild‐type human p53 and mutant p53 (p53R175H) protein expression vectors. The empty vector pcDNA3.1 was used as a control. Relative luciferase activity indicates luciferase activity normalized to the control vectors pcDNA3.1 and pGL4.24. The light blue line denotes the identified p53 BSs scanned with JASPAR p53 transcription factor (TF)‐binding profiles.

Article Snippet: p53‐knockout mice ( p53 −/− ; BRC no.: RBRC00107) with C57BL/6 background were provided by RIKEN BioResource Center.

Techniques: Binding Assay, Expressing, Transfection, Negative Control, Comparison, Mutagenesis, Two Tailed Test, Modification, Construct, Luciferase, Plasmid Preparation, Control, Activity Assay

Journal:

Article Title: The Role of p53 in Bleomycin-Induced DNA Damage in the Lung

doi:

Figure Lengend Snippet: Ki-67 labeling index in the bronchi (top), alveoli (second panel), small intestinal crypts (third panel), and villi (bottom). Solid line, wild-type mice; dashed line, p53 knockout mice.

Article Snippet: Age- and sex-matched C57/BL6 mice homozygously deficient for p53 (p53 knockout mice) 27 were obtained from Immuno-Biological Laboratories (Gunma, Japan).

Techniques: Labeling, Knock-Out

Journal:

Article Title: The Role of p53 in Bleomycin-Induced DNA Damage in the Lung

doi:

Figure Lengend Snippet: Histological appearance of the lung from a wild-type (A) and p53 knockout (B) mouse at 3 days after the treatment. In both the wild-type and p53 knockout mice, a few inflammatory cell infiltrates composed of neutrophils and mononuclear cells were observed around bronchi and small blood vessels and within alveolar spaces. No cells exhibiting apoptotic characteristics were seen in either bronchi or alveoli of either group of mice (insets). Hematoxylin and eosin; original magnification, ×200 (insets, ×400).

Article Snippet: Age- and sex-matched C57/BL6 mice homozygously deficient for p53 (p53 knockout mice) 27 were obtained from Immuno-Biological Laboratories (Gunma, Japan).

Techniques: Knock-Out

Journal:

Article Title: The Role of p53 in Bleomycin-Induced DNA Damage in the Lung

doi:

Figure Lengend Snippet: A: DNA electrophoresis of the lung and small intestine from wild-type mice. Lane 1, marker; Lanes 2–9, lung (control and at 2 hours to 7 days); Lane 10 , small intestine at 12 hours. DNA laddering was observed in the small intestine at 12 hours after the treatment, but not in the lung at any time point. B: Apoptotic index in the bronchi (top), alveoli (second panel), small intestinal crypts (third panel), and villi (bottom) of the wild-type (solid line) and p53 knockout (dashed line) mice. C and D: Histological appearance of the small intestine of a wild-type (C) and p53 knockout (D) mouse; left, crypts at 12 hours; right, villi at 3 days after the treatment. Cells with typical apoptotic characteristics were observed not only in the crypts but also in the villi of both groups (arrows). Hematoxylin and eosin; original magnification, ×400.

Article Snippet: Age- and sex-matched C57/BL6 mice homozygously deficient for p53 (p53 knockout mice) 27 were obtained from Immuno-Biological Laboratories (Gunma, Japan).

Techniques: Nucleic Acid Electrophoresis, Marker, DNA Laddering, Knock-Out

Journal:

Article Title: The Role of p53 in Bleomycin-Induced DNA Damage in the Lung

doi:

Figure Lengend Snippet: Expression of p53 and p21waf1/cip1 in the lung. A: Western blot analysis for p53 and p21waf1/cip1 in the wild-type and p53 knockout mice. Expression of p53 and p21waf1/cip1 was observed in the wild-type mice after the treatment, whereas the expression of these proteins was not detected in the p53 knockout mice throughout the experimental period. B and C: Immunohistochemistry for p53 (B) and p21waf1/cip1 (C) in a wild-type mouse at 12 hours after the treatment. Numerous positive nuclei were observed in the bronchial epithelia and alveoli. B, immunofluorescent staining; original magnification, ×400. C, immunoperoxidase staining; original magnification, ×400.

Article Snippet: Age- and sex-matched C57/BL6 mice homozygously deficient for p53 (p53 knockout mice) 27 were obtained from Immuno-Biological Laboratories (Gunma, Japan).

Techniques: Expressing, Western Blot, Knock-Out, Immunohistochemistry, Staining, Immunoperoxidase Staining

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: Body and gross organ weights (mean ± SEM) on postnatal day 73 or 77. p53−/− rats have similar body weights (A), but significantly lower testis (B, C) and epididymis (D, E) weights, as well as organ:body weight ratios (C,E), compared to p53+/+. *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001 by either two-tailed t-test (testis and epididymis weight) or Mann-Whitney U test (body weight) (n = 5 rats). Left and right testis and epididymis weights from each animal were averaged and the average was analyzed as a single biological replicate.

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: Two Tailed Test, MANN-WHITNEY

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: p53−/− rats have higher rates of atrophy and other testicular pathology than p53+/+. p53+/+ and p53−/− rat testis sections were stained with H&E. Histological images show a normal seminiferous tubule in a p53+/+ testis (A), as well as examples of atrophy (B), a large vacuole (C, arrow), and germ cell sloughing (D) in p53−/− testes. Scale bar = 60 μm. Arrowheads in B identify spermatogonia in the atrophic tubule. p53−/− testes display significantly increased levels of seminiferous tubule atrophy (E) and large vacuoles (F), but not sloughing (G), compared to the wild-type. When all abnormalities were considered together, p53−/− rats also had a significantly higher rate than wild-type (H). Mean ± SEM reported in E–H. Scored 51–73 tubules per left testis from each animal (mean = 57.3 tubules). *p < 0.05 by the Mann-Whitney U test (atrophy, vacuoles, all) or t-test (sloughing) (n = 5 rats).

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: Staining, MANN-WHITNEY

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: p53−/− rats have reduced seminiferous tubule diameter and spermatid release, relative to p53+/+. Representative images of stage VII/VIII seminiferous tubules in a p53+/+ testis (A) and a p53−/− testis (B), showing reduced numbers of elongate spermatids releasing in p53−/− testes. Representative minor axis seminiferous tubule diameters measure 292.2 μm and 265.4 μm, respectively. Scale bar = 100 μm. Seminiferous tubule diameter was measured for 51–73 tubules (mean = 57.3 tubules) on H&E-stained left testis sections. The average tubule diameter (mean ± SEM) is lower in p53−/− testes than p53+/+, including both atrophic and normal p53−/− samples (C). **p < 0.01 by t-test (n = 5 rats).

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: Staining

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: p53−/− rats have elevated rates of testicular germ cell death compared to p53+/+. Compared to p53+/+, p53−/− rat testis exhibits significantly increased rates of TUNEL-positive germ cells (mean ± SEM) (A), particularly TUNEL+ spermatocytes. 33–77 tubules were scored per histological section of left testis from each animal (mean = 59.3 tubules). Atrophic tubules were excluded from the analysis. p53−/− testes had a greater number of TUNEL+ germ cells (B) and spermatogonia (C) per tubule than p53+/+ testes. The mean number of spermatogonia among TUNEL-positive cells trended higher in p53+/+ testes (not significant). ***, adjusted p < 0.001, ** adjusted p < 0.01 by multiple t-tests with Holm-Sidak multiple test correction. *adjusted p < 0.05 by Mann-Whitney U-test (n = 5 p53+/+ and 4 p53−/− rats). In p53+/+ rats (E–H), TUNEL+ spermatogonia (arrows) were frequently detected in stages I–VI (E) and XII–XIV (H). In p53−/− rat testes (I–L), TUNEL+ spermatogonia (arrows) were also detected, predominantly in stages I–VI (I) and XII–XIV (L). TUNEL+ spermatocytes were prevalent in all stages except IX–XI (K). Counterstained with methyl green. Scale bar = 60 μm.

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: TUNEL Assay, MANN-WHITNEY

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: Cleaved caspase-3 is localized predominantly to round spermatids in stages VII–VIII, both in p53+/+ (A) and p53−/− (B) rat testes. In p53+/+, cleaved caspase-3 is moderately intense and clearly expressed in the cytoplasm and perinuclear region. In p53−/−, cleaved caspase-3 intensity is often greater, and includes spermatids with apparent nuclear expression. Scale bar = 50 μm. Hematoxylin counterstain.

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: Expressing

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: γH2AX levels do not differ between p53+/+ and p53−/− rat testes. Western blot analysis indicated no significant difference in γH2AX protein quantity (mean ± SEM) between p53+/+ and p53−/− rats (t-test, n = 5 rats) (A). Lanes labeled with “a” are at least 50% atrophic. Red boxes correspond with atrophic samples labeled on Western blot. Immunofluorescent images of testes stained for γH2AX (red) and Hoechst counterstain (blue) indicate that the pattern of γH2AX localization is similar in the wild-type testis (B, C, F–H) and non-atrophic p53-knockout testis tubules (D, E, I–K). In stages I–VI and VII–VIII (B–E), γH2AX is present in the sex vesicles of pachytene spermatocytes. During stages IX–XIV (F–K), a large number of γH2AX foci are present in leptotene spermatocytes (IX–XI), becoming more focused in zygotene spermatocytes (XII–XIII), and condensing into the punctate sex vesicle stain in early pachytene spermatocytes (XIV). Spermatogonia do not show evidence of γH2AX presence in either genotype (e.g. white arrowhead in D). Scale bar = 20 μm.

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: Western Blot, Labeling, Staining, Knock-Out

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: GFRA1 staining and spermatogonial proliferation in p53−/− rats. Wild-type and knockout rat seminiferous tubules were isolated and GFRA1 was labeled using indirect immunofluorescence (green), with DAPI nuclear counterstain (blue) (A). Scale bar in A = 50 μm Tubules were examined for proportions of isolated (As), paired (Apr), and longer-chain type A spermatogonia (Aal–4, Aal–8, and Aal–16). There was no significant difference in the number of clusters per mm seminiferous tubule (mean ± SEM, t-test) (B). Red symbol represents >50% atrophic testis. There was also no significant difference in the frequency of any category of cluster (mean ± SEM, multiple t-tests with Holm-Sidak multiple test correction, n = 3–4 rats) (C).

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: Staining, Knock-Out, Isolation, Labeling, Immunofluorescence

Journal: Andrology

Article Title: Spontaneous testicular atrophy occurs despite normal spermatogonial proliferation in a Tp53 knockout rat

doi: 10.1111/andr.12409

Figure Lengend Snippet: Effects of X-radiation in p53−/− and p53+/+ rats. Following 0.5 Gy X-ray exposure (A) and 5 Gy X-ray exposure (B), TUNEL rates are significantly higher in p53−/− than p53+/+ rat testis sections (left testis). 12 h after treatment, 5 Gy X-ray treatment produced TUNEL rates that are statistically indistinguishable by genotype (C, right testis). At 12 h after treatment, TUNEL+ spermatogonia (arrows) are present in both p53+/+ and p53−/− tubules, especially in stages I–VI. However, p53−/− tubules also show significant numbers of TUNEL+ spermatocytes (D, scale bar = 50 μm). The proportion of all TUNEL+ cells comprised by spermatogonia was significantly greater in p53+/+ rats than p53−/− rats at all treatments and time points (E–G). Body weight was not significantly different between genotypes on postnatal day 97–102 (6 weeks after X-ray exposure) (H). Right testis (I) and right epididymis (J) weights in p53−/− rats were significantly lower than those in p53+/+ rats, 6 weeks after 0 Gy control or 0.5 Gy X-ray treatment. In all treatment groups and time points, seminiferous tubule apoptosis is observed in p53−/− rats significantly more frequently than p53+/+ rats, and is observed both in the left testis (3 h time point) and right testis (12 h or 6 week time points) (K). *adjusted p < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001; A–C and H–J data analyzed by multiple t-tests with Holm-Sidak multiple test correction (n = 3–6 rats per group). E and F analyzed by unpaired t-test. G analyzed by Mann-Whitney U-test due to small sample size. H analyzed by two-way ANOVA followed by Sidak’s multiple comparison test (n = 15–17 rats/group). All data reported as mean ± SEM.

Article Snippet: Induction of apoptosis via upregulation of Fas in response to X-radiation is inhibited in the testes of p53 knockout mice ( Hasegawa et al. 1998 ; Embree-Ku et al. 2002 ), which could explain abnormal germ cell accumulation.

Techniques: TUNEL Assay, Produced, Control, MANN-WHITNEY, Comparison