Review

pcdna3 tp53 wt (Addgene inc)

Addgene inc is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Addgene inc pcdna3 tp53 wt
Pcdna3 Tp53 Wt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna3+tp53+wt/pcDNA3+p53+WT+(Plasmid+%2369003)/pmc12533597__sciadv%2Eadx6877_sm-83-61-70
Average 93 stars, based on 43 article reviews

pcdna3 tp53 wt - by Bioz Stars, 2026-09

93/100 stars

Images

Gene Expression:

Article Title: Cooperative role of distinctive TP53 and PTEN combined loss in the peripheral T cell lymphoma–GATA3 molecular subgroup
Article Snippet: In silico pathway analysis: Functional annotation analysis was performed on differentially expressed genes using Gene Set Enrichment Analysis (GSEA), ConsensusPathDB (http://cpdb.molgen.mpg.de/), Ingenuity Pathway analysis [QIAGEN Ingenuity Pathway Analysis (IPA)], and DAVID (https://davidbioinformatics.nih.gov/). .. For GSEA, we used the pre-ranked method from the Broad Institute with parameters set to 1000 gene set permutations and these gene sets were obtained from gene expression data (HG-U133 Plus2 platform (Affymetrix) or RNA-seq data) Chromatin immunoprecipitation (ChIP) 293T cells were transfected with pcDNA TP53 WT expression vector using TransIT-LT1 Transfection Reagent (Mirus, Inc) 48 hours prior to formaldehyde fixation. pcDNA3 TP53 WT was a gift from David Meek (Addgene, Inc). .. The chromatin was sheared to ~500 bp by sonication using a Bioruptor (Diagenode, New York, NY) with sonication cycles consisting of 30 seconds ‘on’ and 30 seconds ‘off’ for 7.5 min. Sonicated chromatin was diluted with ChIP dilution buffer (0.01% SDS, 1.2 mM EDTA, 16.7 mM Tris-HCl, 16.7 mM NaCl, 1.1% Triton X-100, and 0.5 mM PMSF) and 1% was removed prior to immunoprecipitation to serve as input DNA for the downstream qPCR analysis.

RNA Sequencing:

Chromatin Immunoprecipitation:

Transfection:

Expressing:

Image Search Results

Figure 1. Overexpression of TP53 variants and wild-type (WT) constructs selected for functional analysis. (A)—Distribution of TP53 variants analyzed in this study. TAD: transactivation domain, PRD: proline-rich domain, DBD: DNA-binding domain, NLS: nuclear localization signal, TD: tetramerization domain, and BD: basic domain. (B)—TP53 mRNA expression levels for all transfected constructs, including empty vector (EV), p53 wild-type (WT) and p53 variants. (C)—Analysis of transcriptional activation (TA) capacity of TP53 variants. Variant activity is represented in relation to wild-type construction activity. The black bars represent the positive (WT) and negative controls (EV), and the gray bars represent the analyzed variants. p-values < 0.05 were considered statistically significant; (*) p < 0.05; (****) p < 0.0001.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 1. Overexpression of TP53 variants and wild-type (WT) constructs selected for functional analysis. (A)—Distribution of TP53 variants analyzed in this study. TAD: transactivation domain, PRD: proline-rich domain, DBD: DNA-binding domain, NLS: nuclear localization signal, TD: tetramerization domain, and BD: basic domain. (B)—TP53 mRNA expression levels for all transfected constructs, including empty vector (EV), p53 wild-type (WT) and p53 variants. (C)—Analysis of transcriptional activation (TA) capacity of TP53 variants. Variant activity is represented in relation to wild-type construction activity. The black bars represent the positive (WT) and negative controls (EV), and the gray bars represent the analyzed variants. p-values < 0.05 were considered statistically significant; (*) p < 0.05; (****) p < 0.0001.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Over Expression, Construct, Functional Assay, Binding Assay, Expressing, Transfection, Plasmid Preparation, Activation Assay, Variant Assay, Activity Assay

Figure 2. p53 overexpression impact in mRNA relative expression of p53’s target genes. The quantifications were normalized by endogenous housekeeping gene GUSB. The WT conditions were settled = 1 and the mRNA expression of CDKN1A (A), MDM2 (B), GADD45A (C) and PMAIP (D) genes were relativized by WT. The statistical comparisons between the variants were performed with their respective WT conditions (control or 10 Gy). p-values < 0.05 (*) were considered statistically significant; (**) p < 0.005; (***) p < 0.001; and (****) p < 0.0001. UT = untreated.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 2. p53 overexpression impact in mRNA relative expression of p53’s target genes. The quantifications were normalized by endogenous housekeeping gene GUSB. The WT conditions were settled = 1 and the mRNA expression of CDKN1A (A), MDM2 (B), GADD45A (C) and PMAIP (D) genes were relativized by WT. The statistical comparisons between the variants were performed with their respective WT conditions (control or 10 Gy). p-values < 0.05 (*) were considered statistically significant; (**) p < 0.005; (***) p < 0.001; and (****) p < 0.0001. UT = untreated.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Over Expression, Expressing, Control

Figure 3. Analysis of p21 and p-p53 Ser15 protein levels upon DNA damage. (A)—Western blot of p53 (53KDa), p-p53 Ser15 (53KDa), p21 (21KDa) and β-actin (42KDa), in a control or irradiated (10 Gy) conditions. Cellular protein levels of p-p53Ser15 (B) and p21 (C) in SK-OV-3 cell line overexpressing exogenous p53 (WT or variants) were quantified in control and 4 h after ionizing irradiation (10 Gy) conditions. p-values < 0.05 (*) were considered statistically significant. The analyzes were performed from two independent experiments.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 3. Analysis of p21 and p-p53 Ser15 protein levels upon DNA damage. (A)—Western blot of p53 (53KDa), p-p53 Ser15 (53KDa), p21 (21KDa) and β-actin (42KDa), in a control or irradiated (10 Gy) conditions. Cellular protein levels of p-p53Ser15 (B) and p21 (C) in SK-OV-3 cell line overexpressing exogenous p53 (WT or variants) were quantified in control and 4 h after ionizing irradiation (10 Gy) conditions. p-values < 0.05 (*) were considered statistically significant. The analyzes were performed from two independent experiments.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Western Blot, Control, Irradiation

Figure 4. Immunofluorescence analysis of p53 protein levels in p53 WT and K139N or V197M variants after irradiation (10 Gy). (A)—Representative images showing nuclear localization of p53 in p53 WT and K139N or V197M variants; scale bar, 40 µm; (B)—The ratio between Integrated Density (IntDen) and cell number of each condition. The presence of variants decreased the nuclear p53 levels compared to p53 WT. p-values < 0.05 were considered statistically significant; (**) p < 0.005.

Journal: Scientific reports

Article Title: Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li-Fraumeni syndrome.

doi: 10.1038/s41598-024-67810-3

Figure Lengend Snippet: Figure 4. Immunofluorescence analysis of p53 protein levels in p53 WT and K139N or V197M variants after irradiation (10 Gy). (A)—Representative images showing nuclear localization of p53 in p53 WT and K139N or V197M variants; scale bar, 40 µm; (B)—The ratio between Integrated Density (IntDen) and cell number of each condition. The presence of variants decreased the nuclear p53 levels compared to p53 WT. p-values < 0.05 were considered statistically significant; (**) p < 0.005.

Article Snippet: TP53 missense variants were generated using as template the pcDNA3:flag-p53 plasmid containing human wild type (WT) TP53 cDNA (Addgene, #10838).

Techniques: Immunofluorescence, Irradiation

Figure 1. tp53 suppresses embryonal rhabdomyosarcoma (ERMS) tumor initiation. (A) Kaplan–Meier plot showing ERMS tumor initiation in tp53-/- and tp53+/+ fish. (B) Representative images of DsRed-positive zebrafish ERMS. Arrows show tumor location for each fish. All tumor-burdened zebrafish are 10 days old. Scale bar = 0.5 mm. (C) Tumor numbers per zebrafish in tp53-/- and tp53+/+ fish. n = 44 (tp53+/+), n=130 (tp53-/-). (D) Ratio of tumor area to total body area in in tp53-/- and tp53+/+ fish. n = 10. (E) Pie chart showing percentage of tumors found in varying regions of tp53-/- and tp53+/+ fish, showing no significant differences in tumor localization. Head – p=0.25848, trunk – p=0.39532, tail – p=0.92034 (two-tailed two proportions Z-test). (F) Representative H&E staining of zebrafish ERMS tumors. Scale bar = 100 µm.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 1. tp53 suppresses embryonal rhabdomyosarcoma (ERMS) tumor initiation. (A) Kaplan–Meier plot showing ERMS tumor initiation in tp53-/- and tp53+/+ fish. (B) Representative images of DsRed-positive zebrafish ERMS. Arrows show tumor location for each fish. All tumor-burdened zebrafish are 10 days old. Scale bar = 0.5 mm. (C) Tumor numbers per zebrafish in tp53-/- and tp53+/+ fish. n = 44 (tp53+/+), n=130 (tp53-/-). (D) Ratio of tumor area to total body area in in tp53-/- and tp53+/+ fish. n = 10. (E) Pie chart showing percentage of tumors found in varying regions of tp53-/- and tp53+/+ fish, showing no significant differences in tumor localization. Head – p=0.25848, trunk – p=0.39532, tail – p=0.92034 (two-tailed two proportions Z-test). (F) Representative H&E staining of zebrafish ERMS tumors. Scale bar = 100 µm.

Article Snippet: Cloning TP53 wild-type and mutants constructs Wild- type TP53 from both human (Addgene plasmid #69003) or zebrafish (3- day- old embryos cDNA) were amplified by PCR and cloned into pENTR- D- TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one- step Gateway reaction between a Gateway- compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR- D- TOPO plasmid.

Techniques: Two Tailed Test, Staining

Figure 2. tp53 is a potent suppressor of proliferation and to a lesser extent of apoptosis. (A) Representative confocal microscopy images of EdU staining on embryonal rhabdomyosarcoma (ERMS) tumor sections and a plot quantifying the percentage of EdU-positive cells. Average of n = 8–11/primary tumors. White arrows show EdU-positive cells. (B) Representative confocal microscopy images of phospho-histone H3 staining on ERMS tumor sections (scale bar = 100 µm). Total number pHH3-positive cells per single ERMS tumor ×200 confocal image section assessed from n7-9 primary tumors. One the right-most panel is a plot quantifying the total number of pHH3-positive cells per single ERMS section. White arrows show pHH3-positive cells. (C, D) Representative flow cytometry analysis of Annexin V staining of tp53+/+and tp53-/- ERMS tumors, respectively. (E) Quantification of flow cytometry analysis of Annexin V staining. Q1 = pre-necrotic cells, Q2 = late apoptosis + necrotic cells, Q3 = living cells, Q4 = early apoptotic cells. n = 7. ns, not significant, p=0.5926, unpaired t-test.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 2. tp53 is a potent suppressor of proliferation and to a lesser extent of apoptosis. (A) Representative confocal microscopy images of EdU staining on embryonal rhabdomyosarcoma (ERMS) tumor sections and a plot quantifying the percentage of EdU-positive cells. Average of n = 8–11/primary tumors. White arrows show EdU-positive cells. (B) Representative confocal microscopy images of phospho-histone H3 staining on ERMS tumor sections (scale bar = 100 µm). Total number pHH3-positive cells per single ERMS tumor ×200 confocal image section assessed from n7-9 primary tumors. One the right-most panel is a plot quantifying the total number of pHH3-positive cells per single ERMS section. White arrows show pHH3-positive cells. (C, D) Representative flow cytometry analysis of Annexin V staining of tp53+/+and tp53-/- ERMS tumors, respectively. (E) Quantification of flow cytometry analysis of Annexin V staining. Q1 = pre-necrotic cells, Q2 = late apoptosis + necrotic cells, Q3 = living cells, Q4 = early apoptotic cells. n = 7. ns, not significant, p=0.5926, unpaired t-test.

Techniques: Confocal Microscopy, Staining, Flow Cytometry

Figure 3. Human TP53 blocks tumor initiation, growth, and proliferation and increases apoptosis in tp53-/- zebrafish. (A) Kaplan–Meier plot showing embryonal rhabdomyosarcoma (ERMS) tumor initiation in tp53-/- fish with or without p53WT expression. Western blot analysis was performed to assess p53WT expression level in tumors. (B) Representative images of ERMS tumors in tp53-/- fish with or without human TP53WT expression. Tumor-burdened zebrafish are between 15 and 20 days old. Scale bar = 1 mm. (C) Ratio of tumor area to total body area in tp53-/- fish with or without expression of TP53WT. n = 18. (D) Number of tumors per tp53-/- zebrafish with or without expression of TP53WT. ns, not significant. n = 36 (tp53-/-), n = 28 (TP53WT). (E) Pie chart showing site of tumor localization in tp53-/- fish with or without expression of TP53WT showing no statistical differences. Head – p=0.20045, trunk – p=0.42858, tail – p=0.3336. Quantification of proliferation (F) and apoptosis (G) via EdU staining (n = 10) and Annexin V staining (n = 3), respectively, for tumors arising in tp53-/- fish with or without expression of TP53WT.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 3. Human TP53 blocks tumor initiation, growth, and proliferation and increases apoptosis in tp53-/- zebrafish. (A) Kaplan–Meier plot showing embryonal rhabdomyosarcoma (ERMS) tumor initiation in tp53-/- fish with or without p53WT expression. Western blot analysis was performed to assess p53WT expression level in tumors. (B) Representative images of ERMS tumors in tp53-/- fish with or without human TP53WT expression. Tumor-burdened zebrafish are between 15 and 20 days old. Scale bar = 1 mm. (C) Ratio of tumor area to total body area in tp53-/- fish with or without expression of TP53WT. n = 18. (D) Number of tumors per tp53-/- zebrafish with or without expression of TP53WT. ns, not significant. n = 36 (tp53-/-), n = 28 (TP53WT). (E) Pie chart showing site of tumor localization in tp53-/- fish with or without expression of TP53WT showing no statistical differences. Head – p=0.20045, trunk – p=0.42858, tail – p=0.3336. Quantification of proliferation (F) and apoptosis (G) via EdU staining (n = 10) and Annexin V staining (n = 3), respectively, for tumors arising in tp53-/- fish with or without expression of TP53WT.

Techniques: Expressing, Western Blot, Staining

Figure 4. Assigning pathogenicity to two human TP53 sarcoma mutations in the kRASG12D-driven embryonal rhabdomyosarcoma (ERMS) model. (A) Lollipop plot showing the two novel, human p53 mutations P153Δ and C176F, as well as the amino acid sequence alignment for human, mouse, and zebrafish protein. (B) DNA sequencing data from osteosarcoma patient confirming the germline P153Δ mutation, as well as somatic A159V mutation. (C, D) p53 immunohistochemistry staining of p53 in ERMS patient-derived xenograft (PDX) SJRHB00011 expressing p53C176F and osteosarcoma expressing p53P153Δ. (E, F) Representative H&E staining of ERMS PDX expressing the C176F mutation and diagnostic biopsy of osteosarcoma tumor expressing osteosarcoma expressing p53P153Δ showing neoplastic tumor cells with pleomorphic nuclei, irregular chromatin pattern, as well as irregular disorganized trabeculae of unmineralized malignant osteoid (stars). (G) Protein expression of mutant p53 in zebrafish ERMS tumors, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. (H) Kaplan–Meier plot showing tumor initiation in tp53-/- fish with or without expression of mutant TP53. (I) Representative images of tumor localization in tp53-/- fish with or without expression of mutant TP53. Age of zebrafish in panels is 37 d. Scale bar = 1 mm (J) Pie chart showing percentage of tumors found in varying regions of tp53-/- fish with or without expression of mutant TP53. Percentages in red indicate a significant difference to tp53-/- (p=0.0096, two-tailed two proportions Z-test). (K) Quantification of Annexin V staining in tumors arising in tp53-/- fish with or without

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 4. Assigning pathogenicity to two human TP53 sarcoma mutations in the kRASG12D-driven embryonal rhabdomyosarcoma (ERMS) model. (A) Lollipop plot showing the two novel, human p53 mutations P153Δ and C176F, as well as the amino acid sequence alignment for human, mouse, and zebrafish protein. (B) DNA sequencing data from osteosarcoma patient confirming the germline P153Δ mutation, as well as somatic A159V mutation. (C, D) p53 immunohistochemistry staining of p53 in ERMS patient-derived xenograft (PDX) SJRHB00011 expressing p53C176F and osteosarcoma expressing p53P153Δ. (E, F) Representative H&E staining of ERMS PDX expressing the C176F mutation and diagnostic biopsy of osteosarcoma tumor expressing osteosarcoma expressing p53P153Δ showing neoplastic tumor cells with pleomorphic nuclei, irregular chromatin pattern, as well as irregular disorganized trabeculae of unmineralized malignant osteoid (stars). (G) Protein expression of mutant p53 in zebrafish ERMS tumors, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. (H) Kaplan–Meier plot showing tumor initiation in tp53-/- fish with or without expression of mutant TP53. (I) Representative images of tumor localization in tp53-/- fish with or without expression of mutant TP53. Age of zebrafish in panels is 37 d. Scale bar = 1 mm (J) Pie chart showing percentage of tumors found in varying regions of tp53-/- fish with or without expression of mutant TP53. Percentages in red indicate a significant difference to tp53-/- (p=0.0096, two-tailed two proportions Z-test). (K) Quantification of Annexin V staining in tumors arising in tp53-/- fish with or without

Techniques: Sequencing, DNA Sequencing, Mutagenesis, Immunohistochemistry, Staining, Derivative Assay, Expressing, Diagnostic Assay, Control, Two Tailed Test

Figure 5. Expression of TP53P153Δ with kRASG12D in tp53-/- zebrafish results in the initiation of medulloblastomas with a shh gene signature. (A) Heatmap from RNAseq analyses comparing tumors expressing kRASG12D;tp53-/- to kRASG12D; tp53-/-; TP53P153Δ (n = 3/group). A total of 643 genes were selected for the heatmap, with adjusted p-value<0.01 and fold-change >10. (B) Enriched Gene Ontology (GO) Biological Processes (BP) in upregulated genes in p53-/- group (left panel with pink bars) consistent with the expected tissue of origin for kRASG12D;tp53-/- to kRASG12D; tp53-/-; TP53P153Δ (right panel

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 5. Expression of TP53P153Δ with kRASG12D in tp53-/- zebrafish results in the initiation of medulloblastomas with a shh gene signature. (A) Heatmap from RNAseq analyses comparing tumors expressing kRASG12D;tp53-/- to kRASG12D; tp53-/-; TP53P153Δ (n = 3/group). A total of 643 genes were selected for the heatmap, with adjusted p-value<0.01 and fold-change >10. (B) Enriched Gene Ontology (GO) Biological Processes (BP) in upregulated genes in p53-/- group (left panel with pink bars) consistent with the expected tissue of origin for kRASG12D;tp53-/- to kRASG12D; tp53-/-; TP53P153Δ (right panel

Techniques: Expressing

Figure 6. TP53Y220C predisposes to head embryonal rhabdomyosarcoma (ERMS) in zebrafish. (A) Surface representation of p53WT (PDB 2XWR) and p53P153Δ (homology model) showing key residues lining a surface exposed pocket (sticks). The green ovals compare the size and shape of the pocket between the two structures. (B) p53 protein expression levels in tp53-/- fish tumors with or without TP53Y220C, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. (C) Kaplan–Meier plot showing tumor initiation in tp53-/- fish, with or without TP53Y220C. (D, F) Representative images of tp53-/- fish with ERMS tumors, with or without TP53Y220C (GFP-positive). Dashed region outlines the tumor. The zebrafish in (F) are 35 d. Scale bar in (F) 1 mm. (E, G) Representative H&E staining of tumors in tp53-/- fish, with or without TP53Y220C. Scale bar = 100 µm. (H) Pie chart showing localization of tumors expressed as a percentage found in varying regions of in tp53-/- fish with and without TP53Y220C. Percentage in red indicates a significant difference to tp53-/- (p=0.01928, two-tailed two proportions Z-test). (I) Quantification of Annexin V staining in tumors of tp53-/- fish with or without expression of TP53Y220C. n = 3–4. (J) Quantification of EdU staining in tumors of tp53-/- fish with or without expression of TP53Y220C. n = 4–9.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 6. TP53Y220C predisposes to head embryonal rhabdomyosarcoma (ERMS) in zebrafish. (A) Surface representation of p53WT (PDB 2XWR) and p53P153Δ (homology model) showing key residues lining a surface exposed pocket (sticks). The green ovals compare the size and shape of the pocket between the two structures. (B) p53 protein expression levels in tp53-/- fish tumors with or without TP53Y220C, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. (C) Kaplan–Meier plot showing tumor initiation in tp53-/- fish, with or without TP53Y220C. (D, F) Representative images of tp53-/- fish with ERMS tumors, with or without TP53Y220C (GFP-positive). Dashed region outlines the tumor. The zebrafish in (F) are 35 d. Scale bar in (F) 1 mm. (E, G) Representative H&E staining of tumors in tp53-/- fish, with or without TP53Y220C. Scale bar = 100 µm. (H) Pie chart showing localization of tumors expressed as a percentage found in varying regions of in tp53-/- fish with and without TP53Y220C. Percentage in red indicates a significant difference to tp53-/- (p=0.01928, two-tailed two proportions Z-test). (I) Quantification of Annexin V staining in tumors of tp53-/- fish with or without expression of TP53Y220C. n = 3–4. (J) Quantification of EdU staining in tumors of tp53-/- fish with or without expression of TP53Y220C. n = 4–9.

Techniques: Expressing, Control, Staining, Two Tailed Test

Figure 7. kdr downstream of TP53P153Δ predisposes to head embryonal rhabdomyosarcoma (ERMS) in tp53-/- zebrafish. (A) Volcano plot comparing head to trunk ERMS tumors. Genes with adj. p-value<0.01 and fold-change >2 are colored in red (upregulated in head tumor) or blue (downregulated in head tumor). Table adjacent the plot shows the top differentially expressed genes. (B) Western blot showing p53, KDR, and RUNX2 protein expression in bone marrow mesenchymal cells, the osteosarcoma PDX expressing TP53P153Δ, and osteosarcoma cell lines SaOS2 and 143B. (C) Representative KDR

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/elife.68221

Figure Lengend Snippet: Figure 7. kdr downstream of TP53P153Δ predisposes to head embryonal rhabdomyosarcoma (ERMS) in tp53-/- zebrafish. (A) Volcano plot comparing head to trunk ERMS tumors. Genes with adj. p-value<0.01 and fold-change >2 are colored in red (upregulated in head tumor) or blue (downregulated in head tumor). Table adjacent the plot shows the top differentially expressed genes. (B) Western blot showing p53, KDR, and RUNX2 protein expression in bone marrow mesenchymal cells, the osteosarcoma PDX expressing TP53P153Δ, and osteosarcoma cell lines SaOS2 and 143B. (C) Representative KDR

Techniques: Western Blot, Expressing

Schematic of the experimental setup to generate embryonal rhabdomyosarcoma (ERMS) tumors in zebrafish via microinjection of indicated linearized DNA constructs into the one-cell-stage zebrafish embryos generated from in crosses of tp53 -/- or tp53 +/+ zebrafish.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: Schematic of the experimental setup to generate embryonal rhabdomyosarcoma (ERMS) tumors in zebrafish via microinjection of indicated linearized DNA constructs into the one-cell-stage zebrafish embryos generated from in crosses of tp53 -/- or tp53 +/+ zebrafish.

Article Snippet: Wild-type TP53 from both human (Addgene plasmid #69003) or zebrafish (3-day-old embryos cDNA) were amplified by PCR and cloned into pENTR-D-TOPO vector, which was verified by DNA sequencing. rag2:TP53 (human) or rag2:tp53 (zebrafish) plasmids were generated by one-step Gateway reaction between a Gateway-compatible plasmid with the zebrafish rag2 promoter flanked by attR sites and the respective pENTR-D-TOPO plasmid.

Techniques: Microinjection, Construct, Generated

( A ) Kaplan–Meier plot showing ERMS tumor initiation in tp53 -/- and tp53 +/+ fish. ( B ) Representative images of DsRed-positive zebrafish ERMS. Arrows show tumor location for each fish. All tumor-burdened zebrafish are 10 days old. Scale bar = 0.5 mm. ( C ) Tumor numbers per zebrafish in tp53 -/- and tp53 +/+ fish. n = 44 ( tp53 +/+ ), n=130 ( tp53 -/- ). ( D ) Ratio of tumor area to total body area in in tp53 -/- and tp53 +/+ fish. n = 10. ( E ) Pie chart showing percentage of tumors found in varying regions of tp53 -/- and tp53 +/+ fish, showing no significant differences in tumor localization. Head – p=0.25848, trunk – p=0.39532, tail – p=0.92034 (two-tailed two proportions Z -test). ( F ) Representative H&E staining of zebrafish ERMS tumors. Scale bar = 100 µm.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Kaplan–Meier plot showing ERMS tumor initiation in tp53 -/- and tp53 +/+ fish. ( B ) Representative images of DsRed-positive zebrafish ERMS. Arrows show tumor location for each fish. All tumor-burdened zebrafish are 10 days old. Scale bar = 0.5 mm. ( C ) Tumor numbers per zebrafish in tp53 -/- and tp53 +/+ fish. n = 44 ( tp53 +/+ ), n=130 ( tp53 -/- ). ( D ) Ratio of tumor area to total body area in in tp53 -/- and tp53 +/+ fish. n = 10. ( E ) Pie chart showing percentage of tumors found in varying regions of tp53 -/- and tp53 +/+ fish, showing no significant differences in tumor localization. Head – p=0.25848, trunk – p=0.39532, tail – p=0.92034 (two-tailed two proportions Z -test). ( F ) Representative H&E staining of zebrafish ERMS tumors. Scale bar = 100 µm.

Techniques: Two Tailed Test, Staining

( A ) Representative confocal microscopy images of EdU staining on embryonal rhabdomyosarcoma (ERMS) tumor sections and a plot quantifying the percentage of EdU-positive cells. Average of n = 8–11/primary tumors. White arrows show EdU-positive cells. ( B ) Representative confocal microscopy images of phospho-histone H3 staining on ERMS tumor sections (scale bar = 100 µm). Total number pHH3-positive cells per single ERMS tumor ×200 confocal image section assessed from n7-9 primary tumors. One the right-most panel is a plot quantifying the total number of pHH3-positive cells per single ERMS section. White arrows show pHH3-positive cells. ( C, D ) Representative flow cytometry analysis of Annexin V staining of tp53 +/+ and tp53 -/- ERMS tumors, respectively. ( E ) Quantification of flow cytometry analysis of Annexin V staining. Q1 = pre-necrotic cells, Q2 = late apoptosis + necrotic cells, Q3 = living cells, Q4 = early apoptotic cells. n = 7. ns, not significant, p=0.5926, unpaired t -test.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of EdU staining on embryonal rhabdomyosarcoma (ERMS) tumor sections and a plot quantifying the percentage of EdU-positive cells. Average of n = 8–11/primary tumors. White arrows show EdU-positive cells. ( B ) Representative confocal microscopy images of phospho-histone H3 staining on ERMS tumor sections (scale bar = 100 µm). Total number pHH3-positive cells per single ERMS tumor ×200 confocal image section assessed from n7-9 primary tumors. One the right-most panel is a plot quantifying the total number of pHH3-positive cells per single ERMS section. White arrows show pHH3-positive cells. ( C, D ) Representative flow cytometry analysis of Annexin V staining of tp53 +/+ and tp53 -/- ERMS tumors, respectively. ( E ) Quantification of flow cytometry analysis of Annexin V staining. Q1 = pre-necrotic cells, Q2 = late apoptosis + necrotic cells, Q3 = living cells, Q4 = early apoptotic cells. n = 7. ns, not significant, p=0.5926, unpaired t -test.

Techniques: Confocal Microscopy, Staining, Flow Cytometry

( A ) Schematic of the protein structure of p53 with the different domains highlighted with different colors. ( B ) The amino acid sequence alignment for p53 between human (Hs) and zebrafish (Dr- Danio rerio ). The colors highlight domains in ( A ); - indicates gaps; : indicates positive substitutions; * indicates conserved amino acids,. ( C ) Table showing amino acid conservation, positive substitutions, and gaps in the full-length and DNA-binding domain.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Schematic of the protein structure of p53 with the different domains highlighted with different colors. ( B ) The amino acid sequence alignment for p53 between human (Hs) and zebrafish (Dr- Danio rerio ). The colors highlight domains in ( A ); - indicates gaps; : indicates positive substitutions; * indicates conserved amino acids,. ( C ) Table showing amino acid conservation, positive substitutions, and gaps in the full-length and DNA-binding domain.

Techniques: Sequencing, Binding Assay

( A ) Lollipop plot showing common p53 mutations in rhabdomyosarcoma patients as well as the seven most common p53 hot spot mutations across all cancers. ( B ) The amino acid sequence alignment for p53 P153 with other species. The yellow highlight shows the amino acid of P153, the top panel shows the conserved ones, and the bottom five species show the non-conserved ones.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Lollipop plot showing common p53 mutations in rhabdomyosarcoma patients as well as the seven most common p53 hot spot mutations across all cancers. ( B ) The amino acid sequence alignment for p53 P153 with other species. The yellow highlight shows the amino acid of P153, the top panel shows the conserved ones, and the bottom five species show the non-conserved ones.

Techniques: Sequencing

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Techniques: Microinjection, Construct, Generated

( A ) Kaplan–Meier plot showing embryonal rhabdomyosarcoma (ERMS) tumor initiation in tp53 -/- fish with or without p53 WT expression. Western blot analysis was performed to assess p53 WT expression level in tumors. ( B ) Representative images of ERMS tumors in tp53 -/- fish with or without human TP53 WT expression. Tumor-burdened zebrafish are between 15 and 20 days old. Scale bar = 1 mm. ( C ) Ratio of tumor area to total body area in tp53 -/- fish with or without expression of TP53 WT . n = 18. ( D ) Number of tumors per tp53 -/- zebrafish with or without expression of TP53 WT . ns, not significant. n = 36 ( tp53 -/- ), n = 28 ( TP53 WT ). ( E ) Pie chart showing site of tumor localization in tp53 -/- fish with or without expression of TP53 WT showing no statistical differences. Head – p=0.20045, trunk – p=0.42858, tail – p=0.3336. Quantification of proliferation ( F ) and apoptosis ( G ) via EdU staining (n = 10) and Annexin V staining (n = 3), respectively, for tumors arising in tp53 -/- fish with or without expression of TP53 WT .

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Kaplan–Meier plot showing embryonal rhabdomyosarcoma (ERMS) tumor initiation in tp53 -/- fish with or without p53 WT expression. Western blot analysis was performed to assess p53 WT expression level in tumors. ( B ) Representative images of ERMS tumors in tp53 -/- fish with or without human TP53 WT expression. Tumor-burdened zebrafish are between 15 and 20 days old. Scale bar = 1 mm. ( C ) Ratio of tumor area to total body area in tp53 -/- fish with or without expression of TP53 WT . n = 18. ( D ) Number of tumors per tp53 -/- zebrafish with or without expression of TP53 WT . ns, not significant. n = 36 ( tp53 -/- ), n = 28 ( TP53 WT ). ( E ) Pie chart showing site of tumor localization in tp53 -/- fish with or without expression of TP53 WT showing no statistical differences. Head – p=0.20045, trunk – p=0.42858, tail – p=0.3336. Quantification of proliferation ( F ) and apoptosis ( G ) via EdU staining (n = 10) and Annexin V staining (n = 3), respectively, for tumors arising in tp53 -/- fish with or without expression of TP53 WT .

Techniques: Expressing, Western Blot, Staining

( A ) Kaplan–Meier plot showing embryonal rhabdomyosarcoma (ERMS) tumor initiation in tp53 -/- fish (AB WT strain) with or without tp53 . ( B ) Zebrafish p53 expression levels in three tp53 -/- tumors expressed rag2: tp53 along with rag2: kRAS G12D , using AB WT tumor driven by rag2: kRAS G12D as control.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Kaplan–Meier plot showing embryonal rhabdomyosarcoma (ERMS) tumor initiation in tp53 -/- fish (AB WT strain) with or without tp53 . ( B ) Zebrafish p53 expression levels in three tp53 -/- tumors expressed rag2: tp53 along with rag2: kRAS G12D , using AB WT tumor driven by rag2: kRAS G12D as control.

Techniques: Expressing, Control

( A ) Lollipop plot showing the two novel, human p53 mutations P153Δ and C176F, as well as the amino acid sequence alignment for human, mouse, and zebrafish protein. ( B ) DNA sequencing data from osteosarcoma patient confirming the germline P153Δ mutation, as well as somatic A159V mutation. ( C, D ) p53 immunohistochemistry staining of p53 in ERMS patient-derived xenograft (PDX) SJRHB00011 expressing p53 C176F and osteosarcoma expressing p53 P153Δ . ( E, F ) Representative H&E staining of ERMS PDX expressing the C176F mutation and diagnostic biopsy of osteosarcoma tumor expressing osteosarcoma expressing p53 P153Δ showing neoplastic tumor cells with pleomorphic nuclei, irregular chromatin pattern, as well as irregular disorganized trabeculae of unmineralized malignant osteoid (stars). ( G ) Protein expression of mutant p53 in zebrafish ERMS tumors, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. ( H ) Kaplan–Meier plot showing tumor initiation in tp53 -/- fish with or without expression of mutant TP53 . ( I ) Representative images of tumor localization in tp53 -/- fish with or without expression of mutant TP53 . Age of zebrafish in panels is 37 d. Scale bar = 1 mm ( J ) Pie chart showing percentage of tumors found in varying regions of tp53 -/- fish with or without expression of mutant TP53 . Percentages in red indicate a significant difference to tp53 -/- (p=0.0096, two-tailed two proportions Z -test). ( K ) Quantification of Annexin V staining in tumors arising in tp53 -/- fish with or without expression of mutant TP53 . n = 6–7. ( L ) Representative H&E staining of tumors arising in tp53 -/- fish with or without expression of mutant TP53 . Scale bar = 100 µm.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Lollipop plot showing the two novel, human p53 mutations P153Δ and C176F, as well as the amino acid sequence alignment for human, mouse, and zebrafish protein. ( B ) DNA sequencing data from osteosarcoma patient confirming the germline P153Δ mutation, as well as somatic A159V mutation. ( C, D ) p53 immunohistochemistry staining of p53 in ERMS patient-derived xenograft (PDX) SJRHB00011 expressing p53 C176F and osteosarcoma expressing p53 P153Δ . ( E, F ) Representative H&E staining of ERMS PDX expressing the C176F mutation and diagnostic biopsy of osteosarcoma tumor expressing osteosarcoma expressing p53 P153Δ showing neoplastic tumor cells with pleomorphic nuclei, irregular chromatin pattern, as well as irregular disorganized trabeculae of unmineralized malignant osteoid (stars). ( G ) Protein expression of mutant p53 in zebrafish ERMS tumors, with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. ( H ) Kaplan–Meier plot showing tumor initiation in tp53 -/- fish with or without expression of mutant TP53 . ( I ) Representative images of tumor localization in tp53 -/- fish with or without expression of mutant TP53 . Age of zebrafish in panels is 37 d. Scale bar = 1 mm ( J ) Pie chart showing percentage of tumors found in varying regions of tp53 -/- fish with or without expression of mutant TP53 . Percentages in red indicate a significant difference to tp53 -/- (p=0.0096, two-tailed two proportions Z -test). ( K ) Quantification of Annexin V staining in tumors arising in tp53 -/- fish with or without expression of mutant TP53 . n = 6–7. ( L ) Representative H&E staining of tumors arising in tp53 -/- fish with or without expression of mutant TP53 . Scale bar = 100 µm.

Techniques: Sequencing, DNA Sequencing, Mutagenesis, Immunohistochemistry, Staining, Derivative Assay, Expressing, Diagnostic Assay, Control, Two Tailed Test

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Techniques: Microinjection, Construct, Generated

( A ) Tumor numbers per zebrafish expressing no TP53 (tp53 -/- ), or TP53 C176F , or TP53 P153Δ .n = 36 ( tp53 -/- ), n = 58 ( TP53 C176F ), n = 81 ( TP53 P153Δ ). ( B ) Percentage of EdU-positive cells in representative areas of fish tumors expressing either no TP53 (tp53 -/- ), TP53 C176F , or TP53 P153Δ . n = 8–10. ( C ) Total number of pHH3-positive cells per single ERMS tumor ×200 confocal image section assessed from tumors expressing WT TP53, TP53 C176F , or TP53 P153 Δ intp53 -/- zebrafish; n = 3–5 primary tumors. ( D ) Representative images of tumor-burdened adult zebrafish (4–6 months of age) with tumors expressing kRAS G12D + TP53 C176F (DsRed+) treated with either DMSO or ZMC1 over a span of 3 wk. ( E ) Western blot showing p53 expression levels in fish tumors expressing kRAS G12D + TP53 C176F and treated with either DMSO or ZMC1. ( F ) Percentage of Annexin V-positive cells in tumors expressing kRAS G12D + TP53 C176F that were treated with either DMSO or ZMC1. n = 4. ( G ) Ratio of tumor size to total body for in fish tumors expressing kRAS G12D + TP53 C176F , treated with either DMSO or ZMC1. ns, not significant, n = 4. ( H ) Representative images of tumor-burdened zebrafish expressing kRAS G12D (DsRed+) treated with either DMSO or ZMC1 over a span of 3 wk. Scale bar = 2 mm ( I ) Western blot showing p53 expression levels in Rh30 cells (control) and fish tumors expressing kRAS G12D , treated with either DMSO or ZMC1. ( J ) Percentage of Annexin V staining of fish tumors expressing kRAS G12D , treated with either DMSO or ZMC1. ns, not significant. n = 6–7. ( K ) Ratio of tumor size to total body in tumors expressing kRAS G12D , treated with either DMSO or ZMC1. ns, not significant, n = 4.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Tumor numbers per zebrafish expressing no TP53 (tp53 -/- ), or TP53 C176F , or TP53 P153Δ .n = 36 ( tp53 -/- ), n = 58 ( TP53 C176F ), n = 81 ( TP53 P153Δ ). ( B ) Percentage of EdU-positive cells in representative areas of fish tumors expressing either no TP53 (tp53 -/- ), TP53 C176F , or TP53 P153Δ . n = 8–10. ( C ) Total number of pHH3-positive cells per single ERMS tumor ×200 confocal image section assessed from tumors expressing WT TP53, TP53 C176F , or TP53 P153 Δ intp53 -/- zebrafish; n = 3–5 primary tumors. ( D ) Representative images of tumor-burdened adult zebrafish (4–6 months of age) with tumors expressing kRAS G12D + TP53 C176F (DsRed+) treated with either DMSO or ZMC1 over a span of 3 wk. ( E ) Western blot showing p53 expression levels in fish tumors expressing kRAS G12D + TP53 C176F and treated with either DMSO or ZMC1. ( F ) Percentage of Annexin V-positive cells in tumors expressing kRAS G12D + TP53 C176F that were treated with either DMSO or ZMC1. n = 4. ( G ) Ratio of tumor size to total body for in fish tumors expressing kRAS G12D + TP53 C176F , treated with either DMSO or ZMC1. ns, not significant, n = 4. ( H ) Representative images of tumor-burdened zebrafish expressing kRAS G12D (DsRed+) treated with either DMSO or ZMC1 over a span of 3 wk. Scale bar = 2 mm ( I ) Western blot showing p53 expression levels in Rh30 cells (control) and fish tumors expressing kRAS G12D , treated with either DMSO or ZMC1. ( J ) Percentage of Annexin V staining of fish tumors expressing kRAS G12D , treated with either DMSO or ZMC1. ns, not significant. n = 6–7. ( K ) Ratio of tumor size to total body in tumors expressing kRAS G12D , treated with either DMSO or ZMC1. ns, not significant, n = 4.

Techniques: Expressing, Western Blot, Control, Staining

Also shown is expression of zebrafish tp53 across the tumors.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: Also shown is expression of zebrafish tp53 across the tumors.

Techniques: Expressing

( A ) Heatmap from RNAseq analyses comparing tumors expressing kRAS G12D ;tp53 - /- to kRAS G12D ; tp53 -/- ; TP53 P153Δ (n = 3/group). A total of 643 genes were selected for the heatmap, with adjusted p-value<0.01 and fold-change >10. ( B ) Enriched Gene Ontology (GO) Biological Processes (BP) in upregulated genes in p53-/- group (left panel with pink bars) consistent with the expected tissue of origin for kRAS G12D ;tp53 - /- to kRAS G12D ; tp53 -/- ; TP53 P153Δ (right panel with light blue bars). ( C ) Representative images of medulloblastoma tumors expressing kRAS G12D and DsRED (top panel) or GFP (bottom two panels) in tp53 -/- fish with or without human TP53 P153 Δ expression. Tumor-burdened zebrafish are between 30 (Fish 1) and 70 (Fish 2, 3) days old. Scale bar = 1 mm. ( D ) Representative H&E staining of tumors arising in the head region of tp53 -/- fish with or without expression of human TP53 P153Δ . Images between 40 and 60 times magnification. ( E–G ) Representative sections of tumor-burdened zebrafish showing H&E staining ( D, E ) and IHC staining for Sox10 ( F ), and Gfap ( G ) in head tumors expressing kRAS G12D in tp53 -/- fish or kRAS G12D and TP53 P153Δ in tp53 -/- fish. Scale bar = 60 µm ( H ) Gene set enrichment analysis (GSEA) showing the enrichment of medulloblastoma gene signatures from Pomeroy et al., and Kool et al., with our zebrafish brain tumors. Log-fold-change derived from the differential expression analysis between tp53 - /- and tp53 -/- ; TP53 P153Δ was used with GSEA pre-ranked function (GSEA, v4.0.3, Broad Institute, MA).

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Heatmap from RNAseq analyses comparing tumors expressing kRAS G12D ;tp53 - /- to kRAS G12D ; tp53 -/- ; TP53 P153Δ (n = 3/group). A total of 643 genes were selected for the heatmap, with adjusted p-value<0.01 and fold-change >10. ( B ) Enriched Gene Ontology (GO) Biological Processes (BP) in upregulated genes in p53-/- group (left panel with pink bars) consistent with the expected tissue of origin for kRAS G12D ;tp53 - /- to kRAS G12D ; tp53 -/- ; TP53 P153Δ (right panel with light blue bars). ( C ) Representative images of medulloblastoma tumors expressing kRAS G12D and DsRED (top panel) or GFP (bottom two panels) in tp53 -/- fish with or without human TP53 P153 Δ expression. Tumor-burdened zebrafish are between 30 (Fish 1) and 70 (Fish 2, 3) days old. Scale bar = 1 mm. ( D ) Representative H&E staining of tumors arising in the head region of tp53 -/- fish with or without expression of human TP53 P153Δ . Images between 40 and 60 times magnification. ( E–G ) Representative sections of tumor-burdened zebrafish showing H&E staining ( D, E ) and IHC staining for Sox10 ( F ), and Gfap ( G ) in head tumors expressing kRAS G12D in tp53 -/- fish or kRAS G12D and TP53 P153Δ in tp53 -/- fish. Scale bar = 60 µm ( H ) Gene set enrichment analysis (GSEA) showing the enrichment of medulloblastoma gene signatures from Pomeroy et al., and Kool et al., with our zebrafish brain tumors. Log-fold-change derived from the differential expression analysis between tp53 - /- and tp53 -/- ; TP53 P153Δ was used with GSEA pre-ranked function (GSEA, v4.0.3, Broad Institute, MA).

Techniques: Expressing, Staining, Immunohistochemistry, Derivative Assay, Quantitative Proteomics

Unbiased gene set enrichment analysis (GSEA) (GSEApreranked function) comparing gene expression between tp53 - /- to tp53 -/- ; TP53 P153Δ tumors (log-fold-change) were performed with chemical and genetic perturbation (CGP) gene sets, part of MSigDB C2 curated gene set collection.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: Unbiased gene set enrichment analysis (GSEA) (GSEApreranked function) comparing gene expression between tp53 - /- to tp53 -/- ; TP53 P153Δ tumors (log-fold-change) were performed with chemical and genetic perturbation (CGP) gene sets, part of MSigDB C2 curated gene set collection.

Techniques: Gene Expression

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Techniques: Microinjection, Construct, Generated

( A ) Representative images of tp53 -/- fish with head embryonal rhabdomyosarcoma (ERMS) tumors, expressing TP53 P153Δ (DsRED positive). The zebrafish in ( A ) are between 25 and 50 d. Scale bar = 1 mm. ( B ) H&E of the head regions of tp53 -/- fish with head ERMS tumors, expressing TP53 P153Δ . ( C ) H&E stained sections at ×20 magnification of tp53 -/- fish with head ERMS tumors, expressing TP53 P153Δ . ( D ) IHC for myosin heavy chain at ×20 magnification of tp53 -/- fish with head ERMS tumors, expressing TP53 P153Δ . Scale bar = 100 µm.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Representative images of tp53 -/- fish with head embryonal rhabdomyosarcoma (ERMS) tumors, expressing TP53 P153Δ (DsRED positive). The zebrafish in ( A ) are between 25 and 50 d. Scale bar = 1 mm. ( B ) H&E of the head regions of tp53 -/- fish with head ERMS tumors, expressing TP53 P153Δ . ( C ) H&E stained sections at ×20 magnification of tp53 -/- fish with head ERMS tumors, expressing TP53 P153Δ . ( D ) IHC for myosin heavy chain at ×20 magnification of tp53 -/- fish with head ERMS tumors, expressing TP53 P153Δ . Scale bar = 100 µm.

Techniques: Expressing, Staining

Log-fold-change derived from the differential expression analysis between tp53 - /- to tp53 -/- ; TP53 P153Δ was used with GSEApreranked function (GSEA, v4.0.3, Broad Institute, MA).

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: Log-fold-change derived from the differential expression analysis between tp53 - /- to tp53 -/- ; TP53 P153Δ was used with GSEApreranked function (GSEA, v4.0.3, Broad Institute, MA).

Techniques: Derivative Assay, Quantitative Proteomics

( A ) Surface representation of p53 WT (PDB 2XWR) and p53 P153Δ (homology model) showing key residues lining a surface exposed pocket (sticks). The green ovals compare the size and shape of the pocket between the two structures. ( B ) p53 protein expression levels in tp53 -/- fish tumors with or without TP53 Y220C , with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. ( C ) Kaplan–Meier plot showing tumor initiation in tp53 -/- fish, with or without TP53 Y220C . ( D, F ) Representative images of tp53 -/- fish with ERMS tumors, with or without TP53 Y220C (GFP-positive). Dashed region outlines the tumor. The zebrafish in ( F ) are 35 d. Scale bar in ( F ) 1 mm. ( E, G ) Representative H&E staining of tumors in tp53 -/- fish, with or without TP53 Y220C . Scale bar = 100 µm. ( H ) Pie chart showing localization of tumors expressed as a percentage found in varying regions of in tp53 -/- fish with and without TP53 Y220C . Percentage in red indicates a significant difference to tp53 -/- (p=0.01928, two-tailed two proportions Z -test). ( I ) Quantification of Annexin V staining in tumors of tp53 -/- fish with or without expression of TP53 Y220C . n = 3–4. ( J ) Quantification of EdU staining in tumors of tp53 -/- fish with or without expression of TP53 Y220C . n = 4–9.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Surface representation of p53 WT (PDB 2XWR) and p53 P153Δ (homology model) showing key residues lining a surface exposed pocket (sticks). The green ovals compare the size and shape of the pocket between the two structures. ( B ) p53 protein expression levels in tp53 -/- fish tumors with or without TP53 Y220C , with rhabdomyosarcoma (RMS) cell line, Rh30, as a control. ( C ) Kaplan–Meier plot showing tumor initiation in tp53 -/- fish, with or without TP53 Y220C . ( D, F ) Representative images of tp53 -/- fish with ERMS tumors, with or without TP53 Y220C (GFP-positive). Dashed region outlines the tumor. The zebrafish in ( F ) are 35 d. Scale bar in ( F ) 1 mm. ( E, G ) Representative H&E staining of tumors in tp53 -/- fish, with or without TP53 Y220C . Scale bar = 100 µm. ( H ) Pie chart showing localization of tumors expressed as a percentage found in varying regions of in tp53 -/- fish with and without TP53 Y220C . Percentage in red indicates a significant difference to tp53 -/- (p=0.01928, two-tailed two proportions Z -test). ( I ) Quantification of Annexin V staining in tumors of tp53 -/- fish with or without expression of TP53 Y220C . n = 3–4. ( J ) Quantification of EdU staining in tumors of tp53 -/- fish with or without expression of TP53 Y220C . n = 4–9.

Techniques: Expressing, Control, Staining, Two Tailed Test

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Techniques: Microinjection, Construct, Generated

( A ) Tumor numbers per zebrafish tumor expressing either no TP53 (tp53 -/- ) or TP53 Y220C . n = 89 ( tp53 -/- ), n = 54 ( TP53 Y220C ). ( B ) Representative images showing variation in tumor localization in fish expressing mutant TP53 (either DsRed positive or GFP positive). Scale bar = 1 mm. Age of the fish 35–45 d.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Tumor numbers per zebrafish tumor expressing either no TP53 (tp53 -/- ) or TP53 Y220C . n = 89 ( tp53 -/- ), n = 54 ( TP53 Y220C ). ( B ) Representative images showing variation in tumor localization in fish expressing mutant TP53 (either DsRed positive or GFP positive). Scale bar = 1 mm. Age of the fish 35–45 d.

Techniques: Expressing, Mutagenesis

( A ) H&E section of the head and trunk regions of a tp53 -/- ;TP53 Y220C zebrafish with a round blue cell tumor (×40–60 magnification). ( B, C ) H&E stained sections at ×20 magnification of tp53 -/- ;TP53 Y220C zebrafish with a round blue cell tumor. Scale bar = 100 µm.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) H&E section of the head and trunk regions of a tp53 -/- ;TP53 Y220C zebrafish with a round blue cell tumor (×40–60 magnification). ( B, C ) H&E stained sections at ×20 magnification of tp53 -/- ;TP53 Y220C zebrafish with a round blue cell tumor. Scale bar = 100 µm.

Techniques: Staining

Also shown is expression of zebrafish tp53 across the tumors. The wt and tp53 -/- tumors are the same controls from .

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: Also shown is expression of zebrafish tp53 across the tumors. The wt and tp53 -/- tumors are the same controls from .

Techniques: Expressing

( A ) Volcano plot comparing head to trunk ERMS tumors. Genes with adj. p-value<0.01 and fold-change >2 are colored in red (upregulated in head tumor) or blue (downregulated in head tumor). Table adjacent the plot shows the top differentially expressed genes. ( B ) Western blot showing p53, KDR, and RUNX2 protein expression in bone marrow mesenchymal cells, the osteosarcoma PDX expressing TP53 P153Δ , and osteosarcoma cell lines SaOS2 and 143B. ( C ) Representative KDR IHC staining of osteosarcoma PDX expressing p53 P153Δ . Scale bar = 60 µm. ( E ) Schematic demonstrating microinjection into the one-cell-stage zebrafish embryos with the indicated constructs to generate GFP-expressing tumors with either kdr or mitfa mosaically deleted in vivo. ( F ) Kaplan–Meier plot showing ERMS tumor initiation in tp53 -/- fish expression TP53 P153Δ with either mitfa or kdr ablation. ( H ) Representative H&E staining of tumors in tp53 -/- fish or tp53 -/- fish expressing TP53 P153Δ with either mitfa or kdr ablation. Scale bar = 60 µm ( G ) Representative images of tumor localization in tp53 -/- fish with expression of TP53 P153Δ with either mitfa or kdr deletion. Fish in the panels are 30 days old. ( I ) Pie chart showing the percentage of tumors found in varying regions of tp53 -/- fish with expression of TP53 P153Δ with either mitfa or kdr deletion, showing significant differences in the head region. *p=0.0088 (two-tailed two proportions Z -test). ( J ) Example of sequencing from an ERMS tumor with a deletion in a portion of kdr exon 13. Five of five of ERMS tumors analyzed have deletions in kdr .

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Volcano plot comparing head to trunk ERMS tumors. Genes with adj. p-value<0.01 and fold-change >2 are colored in red (upregulated in head tumor) or blue (downregulated in head tumor). Table adjacent the plot shows the top differentially expressed genes. ( B ) Western blot showing p53, KDR, and RUNX2 protein expression in bone marrow mesenchymal cells, the osteosarcoma PDX expressing TP53 P153Δ , and osteosarcoma cell lines SaOS2 and 143B. ( C ) Representative KDR IHC staining of osteosarcoma PDX expressing p53 P153Δ . Scale bar = 60 µm. ( E ) Schematic demonstrating microinjection into the one-cell-stage zebrafish embryos with the indicated constructs to generate GFP-expressing tumors with either kdr or mitfa mosaically deleted in vivo. ( F ) Kaplan–Meier plot showing ERMS tumor initiation in tp53 -/- fish expression TP53 P153Δ with either mitfa or kdr ablation. ( H ) Representative H&E staining of tumors in tp53 -/- fish or tp53 -/- fish expressing TP53 P153Δ with either mitfa or kdr ablation. Scale bar = 60 µm ( G ) Representative images of tumor localization in tp53 -/- fish with expression of TP53 P153Δ with either mitfa or kdr deletion. Fish in the panels are 30 days old. ( I ) Pie chart showing the percentage of tumors found in varying regions of tp53 -/- fish with expression of TP53 P153Δ with either mitfa or kdr deletion, showing significant differences in the head region. *p=0.0088 (two-tailed two proportions Z -test). ( J ) Example of sequencing from an ERMS tumor with a deletion in a portion of kdr exon 13. Five of five of ERMS tumors analyzed have deletions in kdr .

Techniques: Western Blot, Expressing, Immunohistochemistry, Microinjection, Construct, In Vivo, Staining, Two Tailed Test, Sequencing

( A ) Schematic of the strategy to target exons 12 and 13 in kdr that contains the enzymatic catalytic domain. ( B ) Sequence of the targeting guide RNAs targeting exon 12/13 in zebrafish kdr. ( C ) Polyacrylamide gel showing wild type, mutant heteroduplex, and complete deletion DNA bands obtained from a 641 PCR amplicon of the region of exons 12 and 13 of kdr that was targeted for ablation by three guide RNAs from five independent embryonal rhabdomyosarcoma (ERMS) tumors (T1–T5) from the tp53 -/- + TP53 P153Δ + Cas9/gRNA targeting kdr experimental group. The presence of deletions as visualized by small PCR amplicons corresponding to a loss of 334 bp in a 641 bp wild-type PCR product (deletion mutant amplicon = 212 bp). In addition, presence of heteroduplex products can be seen via PAGE, indicating the presence of indels. ( D ) Trace DNA sequencing reads from four independent tumors showing that the gRNA used are active and can introduce deletions in exons 12 and 13 of kdr . Indels were confirmed by cloning PCR products and sequencing individual clones. Please see indels +21 bp, –7 bp at the g1 site, 7 bp deletion at the g2 site, and 25 bp deletion at the g3 site. ( E ) Schematic of the strategy to target exon 2 in mitfa . ( F ) Sequence of the targeting guide RNAs targeting exon 2 in zebrafish mitfa.

Journal: eLife

Article Title: Defining function of wild-type and three patient-specific TP53 mutations in a zebrafish model of embryonal rhabdomyosarcoma

doi: 10.7554/eLife.68221

Figure Lengend Snippet: ( A ) Schematic of the strategy to target exons 12 and 13 in kdr that contains the enzymatic catalytic domain. ( B ) Sequence of the targeting guide RNAs targeting exon 12/13 in zebrafish kdr. ( C ) Polyacrylamide gel showing wild type, mutant heteroduplex, and complete deletion DNA bands obtained from a 641 PCR amplicon of the region of exons 12 and 13 of kdr that was targeted for ablation by three guide RNAs from five independent embryonal rhabdomyosarcoma (ERMS) tumors (T1–T5) from the tp53 -/- + TP53 P153Δ + Cas9/gRNA targeting kdr experimental group. The presence of deletions as visualized by small PCR amplicons corresponding to a loss of 334 bp in a 641 bp wild-type PCR product (deletion mutant amplicon = 212 bp). In addition, presence of heteroduplex products can be seen via PAGE, indicating the presence of indels. ( D ) Trace DNA sequencing reads from four independent tumors showing that the gRNA used are active and can introduce deletions in exons 12 and 13 of kdr . Indels were confirmed by cloning PCR products and sequencing individual clones. Please see indels +21 bp, –7 bp at the g1 site, 7 bp deletion at the g2 site, and 25 bp deletion at the g3 site. ( E ) Schematic of the strategy to target exon 2 in mitfa . ( F ) Sequence of the targeting guide RNAs targeting exon 2 in zebrafish mitfa.

Techniques: Sequencing, Mutagenesis, Amplification, DNA Sequencing, Introduce, Cloning, Clone Assay

Review

pcdna3 tp53 wt (Addgene inc)

Structured Review

Images

Related Articles

Gene Expression:

RNA Sequencing:

Chromatin Immunoprecipitation:

Transfection:

Expressing:

Similar Products

Image Search Results