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Santa Cruz Biotechnology p53 do1
P53 Do1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 96/100, based on 15794 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/p53+do1/p53+Antibody/us12595483-480-33-34
Average 96 stars, based on 15794 article reviews

p53 do1 - by Bioz Stars, 2026-09

96/100 stars

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Proximity Ligation Assay:

Article Title: ESR1 and p53 interactome alteration defines mechanisms of tamoxifen response in luminal breast cancer
Article Snippet: .. Figure 4 Tamoxifen disrupts ESR1-p53 interaction in breast cancer patient tumors ESR1-p53 interaction in tamoxifen-treated versus untreated tumor tissue was assayed by bright-field PLA with primary antibodies against ERα (HC20) and p53 (DO1) (Santa Cruz Biotechnology) on breast cancer patient TMA. (A) Representative images of tumor tissue IHC for ERα & p53 and their corresponding PLA interactions are shown. (B) Quantification of PLA scores for ERα-p53 interaction in tamoxifen-treated vs. untreated tumor is shown on the right panels. ..

Immunohistochemistry:

Membrane:

Article Title: Bryophyllum pinnatum Induces p53-Dependent Apoptosis of Colorectal Cancer Cells via Increased Intracellular ROS and G2/M Cell-Cycle Arrest In Vitro and Validated in Silico by Molecular Docking.
Article Snippet: Chemotherapy, radiotherapy and surgical treatments of cancer having several limitations and toxic side‐effects, have led researchers to focus towards development of alternative natural plant‐based therapeutics that can reduce disease severity.. The present research work is mainly focussed towards identifying molecular mechanisms of apoptosis of colorectal cancer cells (HCT116) by perennial herb Bryophyllum pinnatum leaf‐extract via both in vitro experimentations and in silico analysis.. B. pinnatum leaf extract induced highest cytotoxicity at lowest dose (IC50:0.01 mg/mL) against HCT116 cells with 49.5% (p< 0.0001) cellular death, in comparison to other cancer cell lines.

Incubation:

Control:

Image Search Results

Colloidal blue stain and excised bands for p53 immunoprecipitated from nuclear extracts with inducible empty vector or PADI4

Journal: STAR Protocols

Article Title: Protocol for identification of protein citrullination by immunoprecipitation followed by mass spectrometry

doi: 10.1016/j.xpro.2025.104326

Figure Lengend Snippet: Colloidal blue stain and excised bands for p53 immunoprecipitated from nuclear extracts with inducible empty vector or PADI4

Article Snippet: Mouse monoclonal anti-p53 (DO1) (human specific) (recommended starting dilution for IP, 2 μg per 1 x 10 7 cells. For WB, 1:1000) , Santa Cruz Biotechnology , Cat# sc-126; RRID: AB_628082.

Techniques: Staining, Immunoprecipitation, Plasmid Preparation

Base peak chromatograms of recombinant p53 digested with trypsin (top) and chymotrypsin (bottom) Chymotrypsin, due to its lower cleavage specificity compared to trypsin, generates a larger number of peptide fragments. When used in combination with trypsin digestion, chymotrypsin enhances the overall sequence coverage of the p53 protein.

Journal: STAR Protocols

Article Title: Protocol for identification of protein citrullination by immunoprecipitation followed by mass spectrometry

doi: 10.1016/j.xpro.2025.104326

Figure Lengend Snippet: Base peak chromatograms of recombinant p53 digested with trypsin (top) and chymotrypsin (bottom) Chymotrypsin, due to its lower cleavage specificity compared to trypsin, generates a larger number of peptide fragments. When used in combination with trypsin digestion, chymotrypsin enhances the overall sequence coverage of the p53 protein.

Techniques: Recombinant, Sequencing

Representative MS/MS spectra of citrullinated p53 peptides (A) MS/MS spectrum of a tryptic p53 peptide containing three citrullinated residues at positions 335, 337, and 342. (B) MS/MS spectrum of a chymotryptic p53 peptide confirming two of the three citrullination sites identified in the trypsin digest. Citrullinated residues are highlighted in red. The asterisk (∗) indicates fragment ions exhibiting a neutral loss of −43.01 Da.

Journal: STAR Protocols

Article Title: Protocol for identification of protein citrullination by immunoprecipitation followed by mass spectrometry

doi: 10.1016/j.xpro.2025.104326

Figure Lengend Snippet: Representative MS/MS spectra of citrullinated p53 peptides (A) MS/MS spectrum of a tryptic p53 peptide containing three citrullinated residues at positions 335, 337, and 342. (B) MS/MS spectrum of a chymotryptic p53 peptide confirming two of the three citrullination sites identified in the trypsin digest. Citrullinated residues are highlighted in red. The asterisk (∗) indicates fragment ions exhibiting a neutral loss of −43.01 Da.

Techniques: Tandem Mass Spectroscopy

A. Viability in CANVAS lymphoblastoid lines (LCLs) is significantly reduced after 24 h (left) and 48 h (right) treatment with increasing doses of cisplatin (from 0 to 30 µM). Data are shown as single data points, mean and standard error. Unpaired t-test with Welch correction was performed to compare two groups assuming unequal variance between the two. Experiments were carried out on 5 CANVAS and 5 control LCLs and each condition was replicated at least three times. B. Western blot analysis showing the expression of p53, cleaved Caspase-3 and γH2A.X in LCLs from healthy controls (C1-C2) and CANVAS patients (P1-P2-P3) after 24hr (left) or 48 hr (right) treatment with cisplatin 10 μM. Data are shown as single data points, mean and standard error. Statistical significance was assessed using Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test. C. Dot blot quantification of Pt-d(GpG)-DNA adducts in total DNA extracted from CANVAS (red) and healthy controls (black) LCLs upon 1.5h cisplatin pulse administration. The number of LCLs is reported on the right. Individual data points, mean values ± SEM of three different experiments (n=3) are shown. Unpaired t-test with Welch correction was performed to compare two groups assuming unequal variance between the two. D. On the left, representative immunohistochemistry image showing increased γH2A.X signal in the post-mortem cerebellum of CANVAS patients compared to healthy controls. Signal intensity was highest at the interface between the molecular and granular layers (highlighted). On the right, quantification of the γH2A.X cell density at the Purkinje cells layer and the granular cells layer, expressed as number of γH2A.X positive nuclei per mm 2 . Data are shown as single data points, mean and standard error. Unpaired t-test with Welch correction was performed to assess the statistical significance. Scale bar: 250 μm

Journal: bioRxiv

Article Title: CANVAS causing AAGGG repeat expansions cause tissue-specific reduction in RFC1 expression and increase sensitivity to DNA damage

doi: 10.1101/2025.11.18.688292

Figure Lengend Snippet: A. Viability in CANVAS lymphoblastoid lines (LCLs) is significantly reduced after 24 h (left) and 48 h (right) treatment with increasing doses of cisplatin (from 0 to 30 µM). Data are shown as single data points, mean and standard error. Unpaired t-test with Welch correction was performed to compare two groups assuming unequal variance between the two. Experiments were carried out on 5 CANVAS and 5 control LCLs and each condition was replicated at least three times. B. Western blot analysis showing the expression of p53, cleaved Caspase-3 and γH2A.X in LCLs from healthy controls (C1-C2) and CANVAS patients (P1-P2-P3) after 24hr (left) or 48 hr (right) treatment with cisplatin 10 μM. Data are shown as single data points, mean and standard error. Statistical significance was assessed using Brown-Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post-hoc test. C. Dot blot quantification of Pt-d(GpG)-DNA adducts in total DNA extracted from CANVAS (red) and healthy controls (black) LCLs upon 1.5h cisplatin pulse administration. The number of LCLs is reported on the right. Individual data points, mean values ± SEM of three different experiments (n=3) are shown. Unpaired t-test with Welch correction was performed to compare two groups assuming unequal variance between the two. D. On the left, representative immunohistochemistry image showing increased γH2A.X signal in the post-mortem cerebellum of CANVAS patients compared to healthy controls. Signal intensity was highest at the interface between the molecular and granular layers (highlighted). On the right, quantification of the γH2A.X cell density at the Purkinje cells layer and the granular cells layer, expressed as number of γH2A.X positive nuclei per mm 2 . Data are shown as single data points, mean and standard error. Unpaired t-test with Welch correction was performed to assess the statistical significance. Scale bar: 250 μm

Article Snippet: Membranes were blocked for 1 hour in 6% skimmed milk and incubated overnight at 4°C with 2.5% skimmed milk with the following antibodies recognizing human proteins: anti β-actin (2Q1055) mouse monoclonal (cat. no. sc-58673, 1:1’000, Santa Cruz biotechnology), anti p53 (DO1) mouse monoclonal (cat. no. sc-126, 1:1’000, Santa Cruz Biotechnology), anti-cleaved Caspase 3 (D175), rabbit polyclonal (cat. no. 9661S, 1:1’000, Cell Signaling) and anti γH2A.X (JBW3) mouse monoclonal (cat. no. 05-636, 1:1’000, Sigma).

Techniques: Control, Western Blot, Expressing, Dot Blot, Immunohistochemistry

Reduced MDM2 protein in T47D cells causes reduced chromatin phosphoproteins 53BP1 and MDC1. ( A ) Experimental design workflow of the SILAC analysis (created in BioRender. Harmon, K. (2025) https://BioRender.com/oyd7ala ). Chromatin isolated from a mixture of T47D vector control cells (MDM2-competent) cultured in natural amino acid medium, and T47Dshmdm2 cells (MDM2-depleted) cultured in heavy isotope amino acid medium, was subjected to proteolysis followed by phospho-peptide purification and enrichment. Scatter plot represents the H/L ratio versus abundance of peptides identified by mass spectrometry, with those corresponding to TP53BP1 (magenta), TP53 (blue), MCM2 (green), and MDC1(brown) highlighted. ( B ) Chromatin (5 μg) isolated from T47D vector control, T47Dshmdm2, and T47Dshmdmx cells was subjected to SDS–PAGE/western blot analysis for 53BP1, MDC1, MCM4, lamin A/C, and mtp53. ( C and D ) IF of total 53BP1 (i), phospho-53BP1 ser25 (ii), or phospho- 53BP1 ser1778 (iii) within T47D vector control nuclei [(i) 528, (ii) 549, and (iii) 501], T47Dshmdm2 nuclei [(i) 549,(ii) 520, and (iii) 501], and T47Dshmdmx nuclei [(i) 623, (ii) 376, and (iii) 501]. Confocal images for six fields for each were acquired and the number of 53BP1 foci per nucleus from each cell population indicated above was determined. Representative data ( n = 3) with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Reduced MDM2 protein in T47D cells causes reduced chromatin phosphoproteins 53BP1 and MDC1. ( A ) Experimental design workflow of the SILAC analysis (created in BioRender. Harmon, K. (2025) https://BioRender.com/oyd7ala ). Chromatin isolated from a mixture of T47D vector control cells (MDM2-competent) cultured in natural amino acid medium, and T47Dshmdm2 cells (MDM2-depleted) cultured in heavy isotope amino acid medium, was subjected to proteolysis followed by phospho-peptide purification and enrichment. Scatter plot represents the H/L ratio versus abundance of peptides identified by mass spectrometry, with those corresponding to TP53BP1 (magenta), TP53 (blue), MCM2 (green), and MDC1(brown) highlighted. ( B ) Chromatin (5 μg) isolated from T47D vector control, T47Dshmdm2, and T47Dshmdmx cells was subjected to SDS–PAGE/western blot analysis for 53BP1, MDC1, MCM4, lamin A/C, and mtp53. ( C and D ) IF of total 53BP1 (i), phospho-53BP1 ser25 (ii), or phospho- 53BP1 ser1778 (iii) within T47D vector control nuclei [(i) 528, (ii) 549, and (iii) 501], T47Dshmdm2 nuclei [(i) 549,(ii) 520, and (iii) 501], and T47Dshmdmx nuclei [(i) 623, (ii) 376, and (iii) 501]. Confocal images for six fields for each were acquired and the number of 53BP1 foci per nucleus from each cell population indicated above was determined. Representative data ( n = 3) with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Multiplex sample analysis, Isolation, Plasmid Preparation, Control, Cell Culture, Purification, Mass Spectrometry, SDS Page, Western Blot

53BP1 in breast cancer cells interacts with both mtp53 and MDM2. ( A ) Relative abundance of mtp53, MDM2, MDMX, 53BP1, and MDC1 within whole cell extracts (20 μg) prepared from T47D (L194F) and MDA-MB-231 (R280K) cell lines determined by SDS–PAGE/western blot analysis. ( B–D ) Association of mtp53, MDM2, and 53BP1 in vivo measured using the PLA. PLA analyses of mtp53-53BP1 (panel B), MDM2-mtp53 (panel C), and MDM2-53BP1 (panel D) within T47D and MDA-MB-231 cells were performed as described in the “Materials and methods” section; primary antibodies are PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 3 for T47D, n = 2 for MDA-MB-231). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: Panel B vector (T47D = 276; MDA-MB-231 = 108), shmdmx (T47D = 239; MDA-MB-231 = 128), and shmdm2 (T47D = 310; MDA-MB-231 = 106); Panel C vector (T47D = 92; MDA-MB-231 = 104), shmdmx (T47D = 108; MDA-MB-231 = 131), and shmdm2 (T47D = 101; MDA-MB-231 = 136); Panel D vector (T47D = 761; MDA-MB-231 = 129), shmdmx (T47D = 529; MDA-MB-231 = 142), and shmdm2 (T47D = 964; MDA-MB-231 = 163). **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: 53BP1 in breast cancer cells interacts with both mtp53 and MDM2. ( A ) Relative abundance of mtp53, MDM2, MDMX, 53BP1, and MDC1 within whole cell extracts (20 μg) prepared from T47D (L194F) and MDA-MB-231 (R280K) cell lines determined by SDS–PAGE/western blot analysis. ( B–D ) Association of mtp53, MDM2, and 53BP1 in vivo measured using the PLA. PLA analyses of mtp53-53BP1 (panel B), MDM2-mtp53 (panel C), and MDM2-53BP1 (panel D) within T47D and MDA-MB-231 cells were performed as described in the “Materials and methods” section; primary antibodies are PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 3 for T47D, n = 2 for MDA-MB-231). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: Panel B vector (T47D = 276; MDA-MB-231 = 108), shmdmx (T47D = 239; MDA-MB-231 = 128), and shmdm2 (T47D = 310; MDA-MB-231 = 106); Panel C vector (T47D = 92; MDA-MB-231 = 104), shmdmx (T47D = 108; MDA-MB-231 = 131), and shmdm2 (T47D = 101; MDA-MB-231 = 136); Panel D vector (T47D = 761; MDA-MB-231 = 129), shmdmx (T47D = 529; MDA-MB-231 = 142), and shmdm2 (T47D = 964; MDA-MB-231 = 163). **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Techniques: SDS Page, Western Blot, In Vivo, Plasmid Preparation

The MDM2–53BP1 interaction is promoted by the mtp53 C-terminus. ( A ) The C-terminus of mtp53 R273H within MDA-MB-468 was modified using CRISPR–Cas9 to create the cell line MDA-MB-468 R273Hfs347Δ360-393 (termed G6; mtp53 derivative R273HΔC). ( B ) Relative protein levels of 53BP1, MDM2, and mtp53 within MDA-MB-468 (25, 12.5, 6.25, and 3.125 μg) and G6 (25 μg) cell lines was examined by SDS–PAGE/western blot analysis. ( C ) Loss of mtp53 C-terminus disrupts mtp53 co-IP with MDM2. Extracts from MDA-MB-468 and G6 cell lines were incubated with either normal mouse IgG (negative control) or anti-MDM2 antibodies 4B2 (lanes 1–7) or SMP14 (lanes 8–14), and IP reactions were examined for MDM2 and mtp53 by western blot analysis. Lanes 1–7: 4B2 IP reactions from 800 μg of extract (input); lanes contain 10% of total IP and 2% of input. Lanes 8–14: SMP14 IP reactions from 1600 μg of extract; lanes contain 12.5% each IP and 0.5% of input. Lanes labeled 2× (lane 7 for the 4B2 IPs and lane 14 for the SMP14 IPs) contain twice the amount of the G6 extract MDM2 IP; a lighter exposure of mtp53 input is presented due to the vast excess of mtp53 compared to MDM2 within the MDA-MB-468 cell lines. ( D–F ) PLA analysis of mtp53–53BP1 (panel D), MDM2–mtp53 (panel E), and MDM2–53BP1 (panel F) in MDA-MB-468 and G6. PLA analysis of the indicated proteins was measured using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 2A9 antibodies. Confocal images for 3–5 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 2). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (D) R273H = 120; R273HΔC = 144; (E) R273H = 131; R273HΔC = 159; (F) R273H = 102; R273HΔC = 138. **** Indicates a p-value less than or equal to 0.0001.

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2–53BP1 interaction is promoted by the mtp53 C-terminus. ( A ) The C-terminus of mtp53 R273H within MDA-MB-468 was modified using CRISPR–Cas9 to create the cell line MDA-MB-468 R273Hfs347Δ360-393 (termed G6; mtp53 derivative R273HΔC). ( B ) Relative protein levels of 53BP1, MDM2, and mtp53 within MDA-MB-468 (25, 12.5, 6.25, and 3.125 μg) and G6 (25 μg) cell lines was examined by SDS–PAGE/western blot analysis. ( C ) Loss of mtp53 C-terminus disrupts mtp53 co-IP with MDM2. Extracts from MDA-MB-468 and G6 cell lines were incubated with either normal mouse IgG (negative control) or anti-MDM2 antibodies 4B2 (lanes 1–7) or SMP14 (lanes 8–14), and IP reactions were examined for MDM2 and mtp53 by western blot analysis. Lanes 1–7: 4B2 IP reactions from 800 μg of extract (input); lanes contain 10% of total IP and 2% of input. Lanes 8–14: SMP14 IP reactions from 1600 μg of extract; lanes contain 12.5% each IP and 0.5% of input. Lanes labeled 2× (lane 7 for the 4B2 IPs and lane 14 for the SMP14 IPs) contain twice the amount of the G6 extract MDM2 IP; a lighter exposure of mtp53 input is presented due to the vast excess of mtp53 compared to MDM2 within the MDA-MB-468 cell lines. ( D–F ) PLA analysis of mtp53–53BP1 (panel D), MDM2–mtp53 (panel E), and MDM2–53BP1 (panel F) in MDA-MB-468 and G6. PLA analysis of the indicated proteins was measured using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 2A9 antibodies. Confocal images for 3–5 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 2). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (D) R273H = 120; R273HΔC = 144; (E) R273H = 131; R273HΔC = 159; (F) R273H = 102; R273HΔC = 138. **** Indicates a p-value less than or equal to 0.0001.

Techniques: Modification, CRISPR, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Incubation, Negative Control, Labeling

$The MDM2-mtp53 and MDM2–53BP1 interactions are Nutlin 3a sensitive. ( A ) Western blot analysis of whole cell extracts (10 μg) from MCF7 and T47D cell line populations treated for the indicated time with either vehicle or 10 μM Nutlin 3a (labeled N3a or “+” in graphs) for the indicated proteins. ( B ) Nutlin 3a does not inhibit T47D cell cycle progression. Twenty-four hour post-treatment with either vehicle or 10 μM Nutlin 3a, cells were labeled with EdU for 20 min and assayed for Cyclin A2 by immunofluorescence. Confocal images from at least three fields were acquired and the number of EdU + and Cyclin A + nuclei were quantified (tabulated in S4, panel C) in each population of vehicle-treated [vector = 289; shmdmx = 278; shmdm2 = 249] and Nutlin 3a-treated [vector = 227; shmdmx = 283; shmdm2 = 245] cells. The S/G2 fraction (total Cyclin A + nuclei) for vehicle-treated: vector = 33.6%, shmdmx = 37.8%, shmdm2 = 36.8%; Nutlin 3a-treated: vector = 38.3%, shmdmx = 27.0%, shmdm2 = 29.4%. ( C and D ) Nutlin 3a disrupts mtp53–MDM2 and 53BP1–MDM2 PLA foci in T47D. Twenty-four hour post-treatment with vehicle or 10 μM Nutlin 3a cell populations PLA interactions were measured between MDM2–mtp53 (panel C) and MDM2–53BP1 (panel D) using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2 primary antibodies. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus from each cell population was determined ( n = 2 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the materials and methods from the indicated number of cells in panel: (C) vehicle-treated [vector = 328; shmdmx = 298; shmdm2 = 249], Nutlin 3a-treated [vector = 323; shmdmx = 364; shmdm2 = 285] and (D) vehicle-treated [vector = 543; shmdmx = 548; shmdm2 = 455], Nutlin 3a-treated [vector = 610; shmdmx = 545; shmdm2 = 618]. **** Indicates a p-value less than or equal to 0.0001.$

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2-mtp53 and MDM2–53BP1 interactions are Nutlin 3a sensitive. ( A ) Western blot analysis of whole cell extracts (10 μg) from MCF7 and T47D cell line populations treated for the indicated time with either vehicle or 10 μM Nutlin 3a (labeled N3a or “+” in graphs) for the indicated proteins. ( B ) Nutlin 3a does not inhibit T47D cell cycle progression. Twenty-four hour post-treatment with either vehicle or 10 μM Nutlin 3a, cells were labeled with EdU for 20 min and assayed for Cyclin A2 by immunofluorescence. Confocal images from at least three fields were acquired and the number of EdU + and Cyclin A + nuclei were quantified (tabulated in S4, panel C) in each population of vehicle-treated [vector = 289; shmdmx = 278; shmdm2 = 249] and Nutlin 3a-treated [vector = 227; shmdmx = 283; shmdm2 = 245] cells. The S/G2 fraction (total Cyclin A + nuclei) for vehicle-treated: vector = 33.6%, shmdmx = 37.8%, shmdm2 = 36.8%; Nutlin 3a-treated: vector = 38.3%, shmdmx = 27.0%, shmdm2 = 29.4%. ( C and D ) Nutlin 3a disrupts mtp53–MDM2 and 53BP1–MDM2 PLA foci in T47D. Twenty-four hour post-treatment with vehicle or 10 μM Nutlin 3a cell populations PLA interactions were measured between MDM2–mtp53 (panel C) and MDM2–53BP1 (panel D) using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2 primary antibodies. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus from each cell population was determined ( n = 2 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the materials and methods from the indicated number of cells in panel: (C) vehicle-treated [vector = 328; shmdmx = 298; shmdm2 = 249], Nutlin 3a-treated [vector = 323; shmdmx = 364; shmdm2 = 285] and (D) vehicle-treated [vector = 543; shmdmx = 548; shmdm2 = 455], Nutlin 3a-treated [vector = 610; shmdmx = 545; shmdm2 = 618]. **** Indicates a p-value less than or equal to 0.0001.

Techniques: Western Blot, Labeling, Immunofluorescence, Plasmid Preparation

Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. ( A ) Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. 53BP1 was immunoprecipitated from T47D CES as described in the “Materials and methods” section subjected to western blot analysis for 53BP1, MDC1, MDM2, and p53. Lanes are as follows: (1) cytoplasmic extract, (2) CES IP input, (3) IP with IgG, and (4) IP for 53BP1. ( B ) Inhibition of ATM promotes MDM2 activity. Western blot analysis for the indicated proteins within extracts (10 μg) from T47D populations treated for 24 h with either vehicle,10 μM ALRN-6924 (MDM2/X dual inhibitor), 10 μM KU-55933 (ATMi; ATM inhibitor) or both. ( C ) The MDM2 inhibitor ALRN-6924 reduces whereas the ATM inhibitor increases MDC1–53BP1 PLA foci in T47D. Twenty-four hour post treatment with either vehicle,10 μM ALRN-6924, 10 μM KU-55933 (ATMi) or both T47D vector and shmdm2 cells were labeled with EdU for 20 min and then assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Confocal images for several fields were acquired and the number of PLA foci/nucleus from each cell population was determined. Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in: vehicle-treated [vector = 186; shmdm2 = 87], ATMi-treated [vector = 163; shmdm2 = 188], ALRN-6924-treated [vector = 196; shmdm2 = 176], ALRN-6924 + ATMi-treated [vector = 154; shmdm2 = 159]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. ( A ) Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. 53BP1 was immunoprecipitated from T47D CES as described in the “Materials and methods” section subjected to western blot analysis for 53BP1, MDC1, MDM2, and p53. Lanes are as follows: (1) cytoplasmic extract, (2) CES IP input, (3) IP with IgG, and (4) IP for 53BP1. ( B ) Inhibition of ATM promotes MDM2 activity. Western blot analysis for the indicated proteins within extracts (10 μg) from T47D populations treated for 24 h with either vehicle,10 μM ALRN-6924 (MDM2/X dual inhibitor), 10 μM KU-55933 (ATMi; ATM inhibitor) or both. ( C ) The MDM2 inhibitor ALRN-6924 reduces whereas the ATM inhibitor increases MDC1–53BP1 PLA foci in T47D. Twenty-four hour post treatment with either vehicle,10 μM ALRN-6924, 10 μM KU-55933 (ATMi) or both T47D vector and shmdm2 cells were labeled with EdU for 20 min and then assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Confocal images for several fields were acquired and the number of PLA foci/nucleus from each cell population was determined. Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in: vehicle-treated [vector = 186; shmdm2 = 87], ATMi-treated [vector = 163; shmdm2 = 188], ALRN-6924-treated [vector = 196; shmdm2 = 176], ALRN-6924 + ATMi-treated [vector = 154; shmdm2 = 159]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Inhibition, Activity Assay, Plasmid Preparation, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Techniques: Multiplex sample analysis, Isolation, Plasmid Preparation, Control, Cell Culture, Purification, Mass Spectrometry, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Techniques: SDS Page, Western Blot, In Vivo, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Techniques: Modification, CRISPR, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Incubation, Negative Control, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Techniques: Western Blot, Labeling, Immunofluorescence, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Inhibition, Activity Assay, Plasmid Preparation, Labeling

Figure 1. Effect of NaAsO on mRNA and protein levels. (a) Relative expression of TP53 mRNA and protein; (b) relative expression of TLR3 mRNA and protein; (c) relative expression of MDM2 mRNA and protein levels. Data represent n = 3 (mean ± SD) of biological replicates. The analysis was performed using a two-way ANOVA followed by the Bonferroni post hoc test. Significance indicators: ns: not significant, ** p ≤0.005, *** p ≤0.0005.

Journal: International journal of molecular sciences

Article Title: Inorganic Arsenic Induces Elevated p53 Levels with Altered Functionality Impacting the Expression of Toll-like Receptor 3 and Other Target Genes in Immortalized Prostate Epithelial Cells.

doi: 10.3390/ijms26094253

Figure Lengend Snippet: Figure 1. Effect of NaAsO on mRNA and protein levels. (a) Relative expression of TP53 mRNA and protein; (b) relative expression of TLR3 mRNA and protein; (c) relative expression of MDM2 mRNA and protein levels. Data represent n = 3 (mean ± SD) of biological replicates. The analysis was performed using a two-way ANOVA followed by the Bonferroni post hoc test. Significance indicators: ns: not significant, ** p ≤0.005, *** p ≤0.0005.

Article Snippet: Membranes were blocked overnight at 4 ◦C with 5% Blotto non-fat milk (Chem Cruz, Santa Cruz Biotechnology, Dallas, TX, USA), #cat sc-2324) in 1X TPBS and incubated with the primary antibodies anti-TLR3 (1:200, Abcam, Waltham, MA, USA, CB20 0AX), anti-MDM2 (1:500), anti-p53-DO1 (1:1000), or anti-β-actin-HRP (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-126).

Techniques: Expressing

Figure 2. Effect of NaAsO on the expression of p53 target genes (TLR3, MDM2, CDKN1A, BAX, and IGFBP3) in RWPE-1 and CAsE-PE cells. Data are from [18]. Expression was calculated in FPKM. FC: log2 (fold change); adjusted p-value (q-val).

Journal: International journal of molecular sciences

doi: 10.3390/ijms26094253

Figure Lengend Snippet: Figure 2. Effect of NaAsO on the expression of p53 target genes (TLR3, MDM2, CDKN1A, BAX, and IGFBP3) in RWPE-1 and CAsE-PE cells. Data are from [18]. Expression was calculated in FPKM. FC: log2 (fold change); adjusted p-value (q-val).

Techniques: Expressing

Figure 3. RNA-seq analysis of TP53, TLR3, MDM2, and CDKN1A expression in prostate adenocar- cinoma. Data were obtained from OncoDB and normalized by TPM (transcripts per million), with significant values defined as p-value < 0.05.

Journal: International journal of molecular sciences

doi: 10.3390/ijms26094253

Figure Lengend Snippet: Figure 3. RNA-seq analysis of TP53, TLR3, MDM2, and CDKN1A expression in prostate adenocar- cinoma. Data were obtained from OncoDB and normalized by TPM (transcripts per million), with significant values defined as p-value < 0.05.

Techniques: RNA Sequencing, Expressing

Figure 4. Analysis of TP53, TLR3, MDM2, and CDKN1A expression in adrenocortical carcinoma (ACC) (a) and chromophobe kidney carcinoma (KICH) (b) based on RNA-seq data from OncoDB. Expression levels were normalized as TPM (transcripts per million), with significant values defined as p-value < 0.05.

Journal: International journal of molecular sciences

doi: 10.3390/ijms26094253

Figure Lengend Snippet: Figure 4. Analysis of TP53, TLR3, MDM2, and CDKN1A expression in adrenocortical carcinoma (ACC) (a) and chromophobe kidney carcinoma (KICH) (b) based on RNA-seq data from OncoDB. Expression levels were normalized as TPM (transcripts per million), with significant values defined as p-value < 0.05.

Techniques: Expressing, RNA Sequencing

Figure 5. Binding of p53 to the promoter regions of the TLR3 and CDKN1A genes. (a) Diagram showing the location of oligonucleotides in the promoter regions of TLR3 and CDKN1A, respectively. (b) Sequences of oligonucleotides used for amplification of the promoter regions in the ChIP assay (adapted from [14]). (c,d) ChIP results for p53 binding to the promoter regions of TLR3 and CDKN1A in CAsE-PE and HAsE-PE cells compared to their respective controls not exposed to NaAsO (RWPE- 1 and HPrEC). Data are presented as means ± standard deviations from triplicate experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni post hoc test. Significance levels: *** p ≤0.0005.

Journal: International journal of molecular sciences

doi: 10.3390/ijms26094253

Figure Lengend Snippet: Figure 5. Binding of p53 to the promoter regions of the TLR3 and CDKN1A genes. (a) Diagram showing the location of oligonucleotides in the promoter regions of TLR3 and CDKN1A, respectively. (b) Sequences of oligonucleotides used for amplification of the promoter regions in the ChIP assay (adapted from [14]). (c,d) ChIP results for p53 binding to the promoter regions of TLR3 and CDKN1A in CAsE-PE and HAsE-PE cells compared to their respective controls not exposed to NaAsO (RWPE- 1 and HPrEC). Data are presented as means ± standard deviations from triplicate experiments. Statistical analysis was performed using two-way ANOVA followed by Bonferroni post hoc test. Significance levels: *** p ≤0.0005.

Techniques: Binding Assay, Amplification

Figure 6. Sequencing of cDNA from the TP53 gene in CAsE-PE and HAsE-PE cell lines exposed to NaAsO. (a) Schematic representation of the cDNA coding sequence of the TP53 gene, regions and sizes of the fragments amplified from the cDNA of CAsE-PE and HAsE-PE cell lines. (b) Diagram of the p53 protein, highlighting its functional domains: trans-activation domains (TA I, and II), proline- rich region (PPP), DNA binding domain (DBD), oligomerization domain (OD), and amino-terminal trans-activation domain (TD).

Journal: International journal of molecular sciences

doi: 10.3390/ijms26094253

Figure Lengend Snippet: Figure 6. Sequencing of cDNA from the TP53 gene in CAsE-PE and HAsE-PE cell lines exposed to NaAsO. (a) Schematic representation of the cDNA coding sequence of the TP53 gene, regions and sizes of the fragments amplified from the cDNA of CAsE-PE and HAsE-PE cell lines. (b) Diagram of the p53 protein, highlighting its functional domains: trans-activation domains (TA I, and II), proline- rich region (PPP), DNA binding domain (DBD), oligomerization domain (OD), and amino-terminal trans-activation domain (TD).

Techniques: Sequencing, Amplification, Functional Assay, Activation Assay, Binding Assay

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