p53 d01 antibody  (Cell Signaling Technology Inc)

Journal: ACS pharmacology & translational science

Article Title: Covalent Modification of p53 by ( E )-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one.

doi: 10.1021/acsptsci.4c00447

Figure Lengend Snippet: Figure 1. NSC59984 reacts with free thiols via a Michael addition to the α-carbon. (A) Chemical structures of p53-reactivating molecules (NSC59984 and 1), MESNA (Sodium 2-mercaptoethanesulfonate (2)), adduct of Glutathione with NSC59984 (3), adduct of N-acetylcysteine with NSC59984 (4), and adduct of MESNA with NSC59984 (5). Potential sites for nucleophilic attack are labeled as α and β on the NSC59984 structure. The * label in 3, 4, and 5 represents the formation of a new stereogenic center after nucleophilic attack at the α carbon of NSC59984. Molecules 3 and 4 are a mixture of diastereomers, and 5 is a mixture of enantiomers. (B) Proposed scheme describing the reaction between 1 and 2. The * label represents the formation of a new stereogenic center after nucleophilic attack at the α carbon of 1. (C) 1H NMR analysis of time- dependent reaction of model compounds 1 and 2, showing thiol modification. Amplitude was adjusted to reduce signal-to-noise ratio in the stack plot so that specific peaks are more visible. (D) Quantum mechanical calculations of the LUMO for 1 (upper) or NSC59984 (lower) explain the pattern of reactivity.

Article Snippet: TP53 antibody (D01) (Santa Cruz) was diluted 1:50 before incubation, and a secondary antimouse AF647 antibody was used at 5 μg/mL before counter-staining.

Techniques: Labeling, Modification

Journal: ACS pharmacology & translational science

Article Title: Covalent Modification of p53 by ( E )-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one.

doi: 10.1021/acsptsci.4c00447

Figure Lengend Snippet: Figure 3. Modeling of potential structural effects of NSC59984 modification of p53 R248W. (A) Interactions of the Cys124-adduct with the loop of the L1/S3 pocket. In both panels the protein is represented as gray ribbon; side chains of Lys120 and Cys124 are shown as sticks; nitrogen, oxygen, sulfur and hydrogen atoms are colored blue, red, yellow and white, respectively; the carbons of the Cys124-adduct are colored magenta, and the carbons of Lys120 are colored cyan. (B) Cys124 and Cys229 adducts at the parallel interface of p53DBD monomers. The figure was made by superimposing trajectory snapshots onto the C (blue) and A (gray) subunits of a crystal structure of the p53 tetramer bound to DNA.22 The Cys229 and Cys124 adducts, carbons colored purple and magenta, respectively, are part of the top protein monomer. Panel (B-b) shows the Cys229-adduct making two possible hydrogen bonds represented by yellow lines. Panel (B-c) shows the Cys124-adduct making three possible, stabilizing interactions with the loop immediately following S4. The formal +1 charged nitrogen and the partially negatively charged hydroxyl oxygen of Ser166 are indicated by plus and minus signs.

Article Snippet: TP53 antibody (D01) (Santa Cruz) was diluted 1:50 before incubation, and a secondary antimouse AF647 antibody was used at 5 μg/mL before counter-staining.

Techniques: Modification

Journal: ACS pharmacology & translational science

Article Title: Covalent Modification of p53 by ( E )-1-(4-Methylpiperazin-1-yl)-3-(5-nitrofuran-2-yl)prop-2-en-1-one.

doi: 10.1021/acsptsci.4c00447

Figure Lengend Snippet: Figure 4. Effects of biotinylated NSC59984. (A) Analysis of cellular proliferation was determined by fold change in CyQUANT measurement of cellular DNA following treatment with NSC59984-biotin (12 μM) for 72 h over carrier control in CP-A-WT or ESO26-R248W cells. Fold change in carrier-treated cells was normalized to 1. Statistical analysis was performed using a 2-way ANOVA test with Tukey correction. (B) Analysis of cellular proliferation was determined as in (A) following treatment with NSC59984 or NSC59984-biotin (12 μM) for 72 h over carrier control in ESO26-R248W cells. Fold change in carrier-treated cells was normalized to 1. Statistical analysis was performed using a 2-way ANOVA test with Tukey correction. (C) Western blot analysis of p53-biotin protein levels. CP-A-WT or ESO26 cells were treated with NSC59984-biotin (12 μM) for 72 h before cell lysis. Total protein was immunoprecipitated with Streptavidin Mag Sepharose beads and blotted for p53. (D) ESO26-R248W cells were treated with NSC59984-biotin (12 μM) for 2 h. Fixed cells were stained with streptavidin-AF488 (green), and counter stained for DNA (Hoechst, Blue). Images were captured on a Zeiss LSM710 confocal microscope. (E) ESO26-R248W cells were treated with NSC59984 (12 μM) for 12 h as described in panel (D).

Article Snippet: TP53 antibody (D01) (Santa Cruz) was diluted 1:50 before incubation, and a secondary antimouse AF647 antibody was used at 5 μg/mL before counter-staining.

Techniques: CyQUANT Assay, Control, Western Blot, Lysis, Immunoprecipitation, Staining, Microscopy

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A, D, G and J ) mRNA expression of p53, Mdm2, p21 and Bax respectively, in placental lysates at delivery. ( B, E, H, and K ) Protein expression of p53, Mdm2, p21 and Bax, respectively, through densitometry standardized to Myosin Light Chain (MLC) (*p<0.05, **p<0.01, n = 8). ( C, F, I, and L ) Localisation of p53, Mdm2, p21 and Bax respectively, in placental villi by immunohistochemistry. ST = Syncytiotrophoblast. Images counterstained with haematoxylin. Scale bar = 5 µm.

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A ) Transfection of placental explants with fluorescent labelled non-silencing siRNA demonstrating the presence of siRNA in cytotrophoblast (CT) and syncytiotrophoblast (ST). ( B and C ) Quantitative PCR demonstrating significant reductions in p53 and Mdm2 respectively, in explants treated with p53 or Mdm2 siRNA (*p< 0.05, **p<0.01, n = 6). ( D and E ) Western blot densitometry and immunohistochemistry showing a reduction in Mdm2 protein following treatment with Mdm2 siRNA. p53 expression is increased in trophoblast cytoplasm and nuclei in response to Mdm2 siRNA. ( F and G ) TUNEL index and images respectively, showing apoptosis significantly increased in explants exposed to Mdm2 siRNA (*p<0.05). Elevated apoptosis confirmed by M30 immunostaining with examples imaged by electron microscopy. ( H ) Proliferative index and representative images showing significantly reduced proliferation in explants cultured with Mdm2 siRNA (*p<0.05). ( I ) density of syncytial nuclear aggregates (SNA) increased by treatment Mdm2 siRNA (*p<0.05), representative images of control and Mdm2 siRNA (arrow = SNA). Representative images shown (FC = fetal capillary, IVS = intervillous space, CT = cytotrophoblast, ST = syncytiotrophoblast). All scale bars = 10 µm.

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Expressing, TUNEL Assay, Immunostaining, Electron Microscopy, Cell Culture

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A ) Representative Western Blots of control and Nutlin-3 treated normal placental lysates. Densitometry revealed a significant increase in the expression of ( B ) p53, ( C ) Mdm2, ( D ) p21, ( E ) Puma (*p<0.05, n = 5). ( F ) There was no effect on Bax. Co-treatment of BeWo cells with Nutlin-3 (30 µM) and Pifithrin-α (10 µM) reduced caspase-3/7 activity ( G ) and TUNEL staining ( H ) to the level of controls. Representative images of TUNEL staining in ( I ) control, ( J ) Nutlin-3 and ( K ) co-treatment with Nutlin-3 and Pifithrin-α. Blue = DAPI, Green = TUNEL. Scale bar = 50 µm.

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Western Blot, Expressing, Activity Assay, TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A ) Western Blots of Nutlin-3 treated BeWo cell lysates and co-treatment with Nutlin-3 (30 µM) and Pifithrin-α (10 µM) demonstrated no effect upon ( B ) p53, ( C ) Mdm2, ( D ) p21, ( E ) Puma or ( F ) Bax protein expression (n = 5).

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: Co-treatment with Nutlin-3 (30 µM) and Pifithrin-α (10 µM) had no effect on ( A ) p53, ( B ) Mdm2 or ( E ) Bax mRNA expression. Treatment with Nutlin-3 increased ( C ) p21 and ( D ) Puma expression an effect lost by co-treatment with Pifithrin-α (*p<0.05, **p<0.01, n = 5).

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment

doi: 10.1186/s13058-018-1044-5

Figure Lengend Snippet: Chemotherapy-treated patients with tumors harboring TP53 mutation fare equally well or better than patients with TP53 wild-type tumors. ( a ) Position and frequency of the 663 TP53 mutations present in the METABRIC dataset accessed through cBioportal. ( b ) Overall survival curves were created for patients in the METABRIC dataset with TP53 wild-type and mutant tumors from ( b ) all patients; ( c ) those who received chemotherapy (median survival 125 vs 129 months; ( d ) those who received chemotherapy plus radiation (median survival 144 vs 135 months); ( e ) those who received chemotherapy plus radiation but not hormone therapy; ( f ) those who received chemotherapy plus radiation plus hormone therapy. Survival curves were created for patients with TP53 wild-type ( g ) or mutant ( h ) tumors who received chemotherapy plus radiation and no hormone therapy, or chemotherapy plus radiation plus hormone therapy. Overall survival curves were created for patients with TP53 wild-type and mutant tumors from ( i ) PAM50 basal-like tumor cohort that received chemotherapy plus radiation but not hormone therapy; ( j ) the other PAM50 classifications combined [claudin low ( n = 39), HER2 ( n = 50), luminal A ( n = 1), luminal B ( n = 6), normal-like (n = 6)] that received chemotherapy plus radiation but not hormone therapy; ( k ) tumor cohort classified as “triple-negative” in the three gene classifier that received chemotherapy. Statistical differences in survival curves were calculated using both the Wilcoxon test (weighs early events more heavily) and log-rank (Mantel-Cox) tests (weighs events evenly over time). Shown below each survival curve is a table containing the sample size in each arm, the mean +/− standard error of the mean (SEM) and p value (unpaired, two-tailed Student’s t test) for tumor histological grade, tumor stage, tumor size, and Nottingham Prognostic Index

Article Snippet: Western blotting was performed as previously shown [ ] for p53 (D01, Cell Signaling, Danvers, MA, USA) and actin (BA3R, Thermo Fisher Scientific).

Techniques: Mutagenesis, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment

doi: 10.1186/s13058-018-1044-5

Figure Lengend Snippet: TP53 mutation portends worse 5-year overall survival for patients who received hormone therapy, and those who received no chemotherapy. Overall survival curves were created for patients with TP53 wild-type and mutant tumors from cohorts who ( a ) received hormone therapy; ( b ) received hormone therapy but not chemotherapy; ( c ) did not receive chemotherapy. Overall survival curves were created for patients with TP53 wild-type and mutant tumors from cohorts who ( d ) were HER2+; ( e ) were classified as HER2 gain; ( f ) were classified as HER2 gain and received chemotherapy. Statistical differences in survival curves were calculated using both the Wilcoxon test (weighs early events more heavily) and log-rank (Mantel-Cox) tests (weighs events evenly over time). Shown below each survival curve is a table containing the sample size in each arm, the mean +/− SEM and p value (unpaired, two-tailed Student’s t test) for tumor histological grade, tumor stage, tumor size, and Nottingham Prognostic Index

Article Snippet: Western blotting was performed as previously shown [ ] for p53 (D01, Cell Signaling, Danvers, MA, USA) and actin (BA3R, Thermo Fisher Scientific).

Techniques: Mutagenesis, Two Tailed Test

Journal: Breast Cancer Research : BCR

Article Title: Breast cancer survival predicted by TP53 mutation status differs markedly depending on treatment

doi: 10.1186/s13058-018-1044-5

Figure Lengend Snippet: TP53 wild-type, ER+ breast cancer cells made senescent by chemotherapy are sensitive to tamoxifen. ( a ) TP53 wild-type, ER+ cells as indicated were plated in triplicate at 80,000 cells per well in a 24-well plate and then treated with 250 nM doxorubicin for 24 h. Seven days later, 1 μM, 5 μM, or 10 μM tamoxifen (Tam) or ethanol vehicle (ETOH) was added as indicated in the figure, with ( gray bars ) or without ( black bars ) the pan-caspase inhibitor QVD. MTT assay was performed 24 h later. Proliferating cells were plated similarly but treated with tamoxifen the next day. ( b ) MCF-7 cells infected using a lentiviral CRISPR Cas9 system with non-targeting (NT) or TP53 guide RNAs were sorted and then plated and treated with 250 nM doxorubicin as in ( a ). Upper panel : light microscopy images were captured for untreated, proliferating cultures or treated cultures as indicated 8 days following treatment. Scale bar is 100 μm. Lower panels : western blot for p53 ( upper ) and actin ( lower ). ( c ). TP53 mutant, ER+ cell lines as indicated were plated, treated, and MTT assay performed as in ( a ). Statistical analyses of these data are shown in Additional file : Table S3. Data are representative of at least two independent experiments

Article Snippet: Western blotting was performed as previously shown [ ] for p53 (D01, Cell Signaling, Danvers, MA, USA) and actin (BA3R, Thermo Fisher Scientific).

Techniques: MTT Assay, Infection, CRISPR, Light Microscopy, Western Blot, Mutagenesis