Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: E6AP downregulates p53 levels in an HCV Core-dependent manner. ( a – d ) HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the indicated plasmids, for 48 h, followed by Western blotting. The images of the target proteins were cropped from the original images and band intensities were quantified using ImageJ (NIH). Values indicate p53 and E6AP levels relative to the loading control (γ-tubulin).

Article Snippet: Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core stimulates E6AP-mediated proteasomal degradation of p53 but inhibits MDM2-mediated degradation. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the indicated plasmids, for 48 h. For ( a ), HepG2-Core cells stably expressing HCV Core were also included. ( a , b ) Protein levels were measured by Western blot analysis. ( c ) Cells were treated with 50 μM cycloheximide (CHX) for the indicated times before harvesting, followed by Western blotting. Bands of p53 and γ-tubulin were quantified to determine the half-life (t1/2) of p53. The difference in the p53-to-γ-tubulin ratio among samples is shown in a graph. ( d , e ) Cells were treated with 10 μM MG132 for 4 h before harvesting to block further proteasomal degradation. For lanes 5 and 6 in ( e ), cells were treated with Nutlin 3a for 24 h. Total p53 was immunoprecipitated with an anti-p53 antibody and subjected to Western blotting using anti-E6AP, anti-MDM2, anti-HCV Core, and anti-HA antibodies to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed p53, respectively. The input indicates the levels of the designated proteins in the cell lysates. ( f , g ) For mammalian two-hybrid assays, Hep3B cells were transfected with the Gal4 reporter (G5E1b-luc), pSG424-E6AP (or pSG424-MDM2), and pCMV p53-VP16, along with the indicated plasmids, for 48 h, followed by a luciferase assay. For ( f ), cells were treated with 10 μM MG132 for 4 h before harvesting. Luciferase activity from G5E1b-luc was normalized to the β-gal activity measured in the corresponding cell extract. The values show relative luciferase activity compared to the control’s basal level. Results are presented as mean ± SD from four independent experiments ( n = 4). ND, not detected.

Article Snippet: Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes.

Techniques: Transfection, Plasmid Preparation, Expressing, Stable Transfection, Western Blot, Blocking Assay, Immunoprecipitation, Luciferase, Activity Assay

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: E6AP induces ubiquitin-dependent proteasomal degradation of phosphorylated p53 in the presence of HCV Core. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated plasmids, for 48 h. For ( c , d ), cells were treated with 10 μM MG132 for 4 h before harvesting. Band intensities were quantified using ImageJ (NIH). ( a , d ) Protein levels were measured by Western blot analysis. ( b ) Phosphorylated p53 at Ser-15 (pSer-15 p53) in cell lysates was immunoprecipitated with an anti-pSer-15 p53 antibody, followed by Western blotting to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed pSer-15 p53. The input shows the levels of the indicated proteins in cell lysates. ( c ) E6AP or MDM2 were immunoprecipitated with the appropriate antibodies and subjected to Western blotting to detect HCV Core, p53, pSer-15 p53, and pSer-20 p53. The input shows the levels of the indicated proteins in cell lysates. ND, not detected.

Article Snippet: Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core-induced p53 phosphorylation is required for E6AP-mediated proteasomal degradation. ( a ) HepG2 cells transiently transfected with the designated plasmids for 47 h were treated with the indicated concentrations of the ATM inhibitor KU-55933 for 1 h before harvesting, followed by Western blotting. ( b , c ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53 or anti-E6AP antibodies, followed by Western blotting. ( d – f ) For mammalian two-hybrid assays, Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 47 h. Cells were either mock-treated or treated with 10 μM KU-55933 for 1 h before harvesting, followed by a luciferase assay ( n = 4). ND, not detected.

Article Snippet: Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes.

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: p53 phosphorylation is sufficient for E6AP to induce p53 ubiquitination. ( a ) HepG2 cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated amounts of the E6AP expression plasmid, for 48 h. Cells were treated with the designated concentrations of etoposide for 24 h and MG132 for 4 h before harvesting, followed by Western blotting. ( b – d ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53, anti-pSer-15 p53, or anti-E6AP antibodies, followed by Western blotting. ( e – g ) Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 48 h. Cells were either mock-treated or treated with 10 μM etoposide for 24 h before harvesting, followed by a luciferase assay ( n = 4).

Article Snippet: Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: Phosphorylation of p53 at Ser-15 is crucial for E6AP-mediated protein degradation. Hep3B cells were transfected with either wild-type (WT) p53 or p53 mutants with substitutions at Ser-15 and/or Ser-20, along with the indicated plasmids, for 48 h. ( a , b , d ) Protein levels were determined by Western blotting. ( c , e ) Cell lysates were immunoprecipitated with an anti-p53 antibody, and the immunoprecipitates were analyzed by Western blotting.

Article Snippet: Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes.

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core triggers E6AP-dependent ubiquitination of p53 during HCV replication in Huh7D cells. ( a – d ) Huh7D cells were transfected with specific plasmids for 24 h, then infected with HCV for another 24 h. ( a – c ) Protein levels were detected by Western blotting. Myc-p53 indicates p53 from ectopic expression, while Y220C-p53 represents the endogenous form in Huh7D cells. For ( b ), cells were either mock-treated or exposed to the indicated concentrations of Heclin for 12 h before harvesting. ( d ) Cell lysates were immunoprecipitated with an anti-Myc antibody, and the precipitates were analyzed by Western blotting. ( e ) Schematic diagram illustrating how HCV Core regulates proteasomal degradation of p53 mediated by MDM2 and E6AP. Each step is described in the Discussion.

Article Snippet: Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes.

Techniques: Ubiquitin Proteomics, Transfection, Infection, Western Blot, Expressing, Immunoprecipitation

Journal: Cells

Article Title: Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo

doi: 10.3390/cells11223614

Figure Lengend Snippet: AX and AXT cells contain R270C mutant p53. ( a ) AX and AXT cells contain a missense mutation in the DNA-binding domain that occurs as a result of amino acid substitution. ( b ) A comparison of mouse Trp53 and human TP53 sequences at the mutation site. The red text indicates the mutated codon.

Article Snippet: Sheared chromatin (30 μg) was incubated with 10 μL of the anti-p53 antibody (Cell Signaling Technology, #32532) at 4 °C overnight with rotation.

Techniques: Mutagenesis, Binding Assay, Comparison

Journal: Cells

Article Title: Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo

doi: 10.3390/cells11223614

Figure Lengend Snippet: The R270C mutant does not block the function of wild-type p53. ( a ) Immunoblot analysis of p53 expression using an antibody (#ab90363) in the AX and p53-knockout AX cells with or without 0.5 µM doxorubicin for 16 h. KO, knockout; NSB, nonspecific bands; Doxo, doxorubicin. ( b ) The growth of the AX and p53-knockout AX cells. The ratio relative to the value for day 0 was calculated for each datapoint. ( c ) The viability of the AX and p53-knockout AX cells was assessed after 2-day exposure to the indicated concentrations of doxorubicin. The statistical significance between AX cells and the respective knockout cells is shown. ( d , e ) RT and real-time PCR analysis of Cdkn1a ( d ) and Mdm2 ( e ) mRNA in the AX and p53-knockout AX cells treated with or without 0.5 µM doxorubicin for 16 h. The data are normalized to the corresponding levels of Actb mRNA and are shown as the means ± SD of triplicate values. (* p < 0.05; ** p < 0.005).

Article Snippet: Sheared chromatin (30 μg) was incubated with 10 μL of the anti-p53 antibody (Cell Signaling Technology, #32532) at 4 °C overnight with rotation.

Techniques: Mutagenesis, Blocking Assay, Western Blot, Expressing, Knock-Out, Real-time Polymerase Chain Reaction

Journal: Cells

Article Title: Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo

doi: 10.3390/cells11223614

Figure Lengend Snippet: Knockout of wild-type and mutant p53 in AX cells did not prevent tumor progression. ( a , b ) Microscope images of the invasion assay using the AX and p53-knockout AX cells. Representative purple-stained cells are indicated with arrows. The mean number of invasive cells counted in three different fields of each transwell in triplicate experiments is shown in ( b ). ( c ) The schedule of cell inoculation into C57BL/6 SCID mice. ( d ) The dot plot of weight of primary tumors. The horizontal bars and boxed regions show the means and the 95% confidence intervals, respectively. ( e , f ) RT and real-time PCR analysis of GFP mRNA in whole blood ( e ) or a lung ( f ) derived from mice inoculated with the indicated cells. (* p < 0.05; NS, not significant).

Article Snippet: Sheared chromatin (30 μg) was incubated with 10 μL of the anti-p53 antibody (Cell Signaling Technology, #32532) at 4 °C overnight with rotation.

Techniques: Knock-Out, Mutagenesis, Microscopy, Invasion Assay, Staining, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Cells

Article Title: Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo

doi: 10.3390/cells11223614

Figure Lengend Snippet: Knockout of R270C mutant p53 did not affect the invasion ability of AXT cells. ( a ) RT and real-time PCR analysis of Trp53 mRNA in AX and AXT cells. The data are normalized to the corresponding levels of Actb mRNA and are shown as the means ± SD of triplicate values. ( b ) Immunoblot analysis of p53 expression using an antibody (#ab90363) in the AXT and p53-knockout AXT cells. ( c ) The growth of the AXT and p53-knockout AXT cells. The ratio relative to the value for day 0 was calculated for each datapoint. ( d , e ) Flow cytometry analysis of DNA content in the AXT and p53-knockout AXT cells. The size of each fraction of cells is shown in ( e ). ( f ) The viability of the AXT and p53-knockout AXT cells was assessed after 2-day exposure to the indicated concentrations of doxorubicin. ( g , h ) Microscope images of the invasion assay using the AXT and p53-knockout AXT cells. Representative purple-stained cells are indicated with arrows. The mean number of invasive cells counted in three different fields of each transwell in triplicate experiments is shown in ( h ). (* p < 0.05; ** p < 0.005; NS, not significant).

Article Snippet: Sheared chromatin (30 μg) was incubated with 10 μL of the anti-p53 antibody (Cell Signaling Technology, #32532) at 4 °C overnight with rotation.

Techniques: Knock-Out, Mutagenesis, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Flow Cytometry, Microscopy, Invasion Assay, Staining

Journal: Cells

Article Title: Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo

doi: 10.3390/cells11223614

Figure Lengend Snippet: Depletion of mutant p53 did not prevent osteosarcoma progression in vivo. ( a , c ) The schedule of cell inoculation into syngeneic C57BL/6 mice. ( b , d ) The dot plot of weight of primary tumors. The horizontal bars and boxed regions show the means and the 95% confidence intervals, respectively. ( e ) Hematoxylin-eosin (H&E) staining of primary tumors in the mice inoculated with the indicated cells. ( f ) Representative images of the lung metastatic lesions stained with GFP. The arrows indicate lung metastases classified according to whether the major axis was larger than 100 μm. ( g ) The number of metastases present throughout a randomly sliced section was counted. The data represent the mean values from the right lungs of four mice. ( h , i ) RT and real-time PCR analysis of GFP mRNA in whole blood ( h ) or a lung ( i ) derived from the mice inoculated with the indicated cells. (** p < 0.005; NS, not significant).

Article Snippet: Sheared chromatin (30 μg) was incubated with 10 μL of the anti-p53 antibody (Cell Signaling Technology, #32532) at 4 °C overnight with rotation.

Techniques: Mutagenesis, In Vivo, Staining, Real-time Polymerase Chain Reaction, Derivative Assay

Journal: Cells

Article Title: Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo

doi: 10.3390/cells11223614

Figure Lengend Snippet: R270C mutant p53 in AXT cells binds to chromatin. ( a ) A Venn diagram illustrating the genomic overlap of peaks in the active regions of p53, H3K4me3, and H3K9me3. The active regions are defined in the Methods Section. ( b , c ) Density plots of the ChIP-seq reads for p53, H3K4me3, and H3K9me3 at the Cdkn1a ( b ) or Mdm2 ( c ) loci. The data are based on the snapshot images taken from analysis using the IGV software. ( d , e ) ChIP-seq occupancy profiles for p53, H3K4me3, and H3K9me3 at the Ccnd1 ( d ) and Trp53 ( e ) loci. ( f , g ) RT and real-time PCR analysis of the Cdkn1a ( f ) and Mdm2 ( g ) mRNA in the AX, AXT, and p53-knockout AXT cells treated with or without 0.5 µM doxorubicin for 16 h. The data are normalized to the corresponding levels of Actb mRNA and are shown as the means ± SD of triplicate values.

Article Snippet: Sheared chromatin (30 μg) was incubated with 10 μL of the anti-p53 antibody (Cell Signaling Technology, #32532) at 4 °C overnight with rotation.

Techniques: Mutagenesis, ChIP-sequencing, Software, Real-time Polymerase Chain Reaction, Knock-Out

Journal: Cells

Article Title: Depletion of R270C Mutant p53 in Osteosarcoma Attenuates Cell Growth but Does Not Prevent Invasion and Metastasis In Vivo

doi: 10.3390/cells11223614

Figure Lengend Snippet: R270C mutant p53 exhibits a different DNA-binding profile from wild-type p53. ( a ) AO cells harbor wild-type p53. ( b ) RT and real-time PCR analysis of the Cdkn1a and Mdm2 mRNA in AO and AXT cells treated with or without 0.5 µM doxorubicin for 8 h. The data are normalized to the corresponding levels of Actb mRNA and are shown as the means ± SD of triplicate values. ( c ) Immunoblot analysis of p53 expression in AO and AXT cells with or without 0.5 µM doxorubicin for 16 h. ( d ) Map of the Cdkn1a gene locus showing the binding profile of R270C mutant p53 in AXT cells, the transcription start site (+1), and the location of ten amplicons used in real-time qPCR analyses. Numbers indicate the position of the 5’ end of the amplicon relative to the transcription start site. The snapshot image was taken from analysis using the IGV software. ( e ) Screening of the p53 binding levels of wild-type p53 in AO cells treated with or without 0.5 μM of doxorubicin for 8 h. The result of a single experiment is shown. ( f ) DNA binding levels of wild-type p53 or R270C mutant p53 were evaluated by ChIP-qPCR analyses. ChIP assays were performed with cell extracts obtained from AO or AXT cells treated with or without 0.5 μM doxorubicin for 8 h. The results shown are the means ± SE of nine independent PCRs of three independent experiments. (NS, not significant).

Article Snippet: Sheared chromatin (30 μg) was incubated with 10 μL of the anti-p53 antibody (Cell Signaling Technology, #32532) at 4 °C overnight with rotation.

Techniques: Mutagenesis, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Amplification, Software, ChIP-qPCR

Journal: Journal of proteome research

Article Title: Analysis of Stratifin Expression and Proteome Variation in a Rat Model of Acute Lung Injury.

doi: 10.1021/acs.jproteome.4c00980

Figure Lengend Snippet: Figure 6. Western blotting and qRT-PCR analyses of PAI-1, SFN, and p53. (A) Representative Western blot images of the abundance of PAI-1, SFN, and p53-related proteins in the lung tissue extracts from OA-treated rats. (B, D, F) Graphs depicting the quantitation of phosphorylated p53 at Ser15 (pp53-Ser15, B), PAI-1 (Serpine1, D), and SFN (F) by ImageJ software. The abundance of pp53-Ser15 was normalized with that of total p53, and the abundance values of PAI-1 and SFN were normalized with that of β-actin. Data are shown as mean ± SEM (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs 0 h using one-way ANOVA and Dunnett’s posthoc test. (C, E) mRNA levels of PAI-1 (Serpine1, C) and SFN (Sfn, E) in lung tissues of OA-treated rats. mRNA levels were quantified using qRT-PCR and normalized with those of Tbp. Data are shown as mean ± SEM (n = 5 or 6). ***p < 0.001, ****p < 0.0001 vs 0 h using two-way ANOVA and Bonferroni’s posthoc test.

Article Snippet: For detecting each protein, the following rabbit monoclonal antibodies (mAb) or polyclonal antibodies (pAb) were used: rabbit anti-plasminogen activator inhibitor-1 (PAI-1) pAb (#27535, Cell Signaling Technology, Leiden, The Netherlands; 1:1000) rabbit anti-p53 mAb (clone E9B5W [#30313, Cell Signaling Technology; 1:2000]), rabbit anti-phospho-p53 Ser15 pAb (#9284, Cell Signaling Technology; 1:2000), and βactin rabbit pAb (#20536−1-A, Proteintech, Rosemont, IL; 1:5000).

Techniques: Western Blot, Quantitative RT-PCR, Quantitation Assay, Software

Journal: Scientific Reports

Article Title: Regulation of cardiomyocyte DNA damage and cell death by the type 2A protein phosphatase regulatory protein alpha4

doi: 10.1038/s41598-021-85616-5

Figure Lengend Snippet: Regulation of the DNA damage response by α4 protein expression in cardiomyocytes. H9c2 cardiomyocytes were transfected with either 50 nM non-targeting control (NTC) siRNA or 50 nM siRNA specific to rat α4 protein (siα4) for 1–4 days (n = 4) and the KD of α4 protein expression was confirmed by Western analysis using an antibody raised against human α4 ( a ). α4 protein expression was knocked down for 4 days and Western analysis (short and long exposures) was used to probe samples (n = 3) with an antibody specific to phosphorylated S / T QG motifs within ATM/ATR substrates. Arrows indicate substrates whose phosphorylation status (n = 3) was elevated in response to α4 KD ( b ). The expression and phosphorylation (Ser15) of p53 were determined by Western analysis using rodent-specific antibodies raised against mouse p53 protein in samples following 4 days of α4 KD, n = 5 ( c ). The expression of PARP was determined by Western analysis (short and long exposures) following 1–4 days of α4 KD, n = 3. Arrows indicate full length PARP (116 kDa) and cleaved fragments (89 kDa and 24 kDa) ( d ). All data represents data mean values ± SEM and statistical significance vs. non-targeting control (NTC) was determined by a one-tailed Student’s T-test. Expression of β-actin was used to confirm equal protein loading between samples.

Article Snippet: Antibodies to detect Bax (#2772), Bcl-xL (#2762), phosphospecific ATM/ATR substrate (#6966), IGBP1 (α4) (#5699), PARP (#9542), phosphospecific (Ser139) H2AX (#9718), H2AX (#2595), rodent-specific p53 (#32532), rodent-specific phosphospecific (Ser15) p53 (#12571) and V5-tag (#13202) were obtained from Cell Signaling Technology (USA).

Techniques: Expressing, Transfection, Control, Western Blot, Phospho-proteomics, One-tailed Test

Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Exposure to repeated UVB plus heat significantly decreased acetylated p53 levels in keratinocytes. Representative immunohistochemical staining of ex vivo skin either untreated, or exposed to heat, UVB or UVB plus heat, for nuclear DNA (DAPI, blue ), CPD ( red ) and ( a ) phosphorylated SIRT1 (SIRT1-p), ( b ) caspase (casp-3), ( c ) total p53 or ( d ) acetylated p53 (p53-a382). Inset images are an enlarged view of cells positive for CPD and SIRT1 ( red arrows ), CPD and Casp-3 ( orange arrows ), CPD and p53 ( green arrows ) or CPD and p53-a382 ( blue arrows ). Broken lines denote the epidermal/dermal border. All images are at 400X magnification

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Immunohistochemical staining, Staining, Ex Vivo

Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Effect of UVB and/or heat exposure in keratinocytes of the ex vivo skin models or NHEK in vitro

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Ex Vivo

Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Effect of UV and/or heat exposure on p53 downstream gene targets and cell proliferation. a-b Fold change on mRNA expression of BAX, Survivin, ERCC1 or XPC in keratinocytes of the ( a ) skin and ( b ) in vitro, relative to untreated controls. c-d Bar graphs of the percentage mean (+/− SD) of keratinocytes positive for ki67 per field of view either ( c ) in ex vivo skin or ( d ) in vitro. Statistically significant differences are indicated with ** p < 0.001 and/or *** for p -values p < 0.0001

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Ex Vivo

Journal: BMC Dermatology

Article Title: SIRT1 activation mediates heat-induced survival of UVB damaged Keratinocytes

doi: 10.1186/s12895-017-0060-y

Figure Lengend Snippet: Inhibition of SIRT1 in UVB plus heat significantly increased acetylated p53 protein expression and apoptosis of keratinocytes in vitro. a Immunoblot showing levels of total p53, and acetylated p53 protein in UVB, UVB plus heat and UVB plus heat with the SIRT1 inhibitor (Ex-527). b Quantification of protein levels by relative average density standardised by β-actin. c Representative immunohistochemical staining of cells positive for CPD and p53 ( white arrows ), CPD and p53-a382 ( green arrows ) in primary keratinocytes exposed to UVB or UVB plus heat with or without SIRT1 inhibitor Ex-527. All images are at 400X magnification. d Bar graphs of percentage mean (+/− SD) of DNA damaged (CPD), apoptotic, p53 or p53-a382 positive primary keratinocytes exposed to UVB or UVB plus heat with or without Ex-527. Statistically significant differences are indicated with ** for p < 0.001 and/or *** for p -values p < 0.0001 respectively

Article Snippet: The membrane was probed with rabbit monoclonal p53 antibody or p53 acetyl K382 and detected using Clarity Western ECL Blotting Substrate detection kit (Bio-Rad) as per the manufacturer’s instructions.

Techniques: Inhibition, Expressing, In Vitro, Western Blot, Immunohistochemical staining, Staining

Journal: OncoTargets and Therapy

Article Title: Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases

doi: 10.2147/ott.s136785

Figure Lengend Snippet: Figure 1 Expressions of p53 mRNA in (A) MUM-2B, C918, and (B) D78 cells as detected by RT-PCR. Abbreviations: mRNA, messenger RNA; RT-PCR, real-time polymerase chain reaction.

Article Snippet: Primary antibodies used for immunohistochemical evaluation included an antihuman polyclonal p53 antibody (1:500, clone CM1; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: OncoTargets and Therapy

Article Title: Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases

doi: 10.2147/ott.s136785

Figure Lengend Snippet: Figure 2 Expression of p53 protein in MUM-2B, C918, and D78 cells as detected by Western blot.

Article Snippet: Primary antibodies used for immunohistochemical evaluation included an antihuman polyclonal p53 antibody (1:500, clone CM1; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.).

Techniques: Expressing, Western Blot

Journal: OncoTargets and Therapy

Article Title: Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases

doi: 10.2147/ott.s136785

Figure Lengend Snippet: Figure 4 The cell invasion ability was measured by a transwell assay at 48 hours after p53 overexpression in cells. Note: Magnification ×200.

Article Snippet: Primary antibodies used for immunohistochemical evaluation included an antihuman polyclonal p53 antibody (1:500, clone CM1; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.).

Techniques: Transwell Assay, Over Expression

Journal: OncoTargets and Therapy

Article Title: Evaluation of p53 gene expression and prognosis characteristics in uveal melanoma cases

doi: 10.2147/ott.s136785

Figure Lengend Snippet: Figure 3 Expression of p53 protein in pathological tissues as detected by immunohistochemistry method (400×).

Article Snippet: Primary antibodies used for immunohistochemical evaluation included an antihuman polyclonal p53 antibody (1:500, clone CM1; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.).

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Sulforaphane downregulates AR-mediated ROS/p53 signaling in HG-stimulated human platelets. (A–D) Washed human platelets were pre-incubated with SFN (5, 10, or 20 μM) or vehicle control for 40 min, followed by stimulation with NG or HG for additional 90 min. (A,B) Intraplatelet ROS levels were measured by flow cytometry. (C,D) Platelets were lysed and phosphorylation of p38α MAPK (C) and p53 (D) were determined by Western blotting. (E–J) Washed human platelets were pre-incubated with SFN (20 μM) with or without a ROS scavenger NAC (500 μM) (E–G) or ARI (10 μM) (H–J) for 40 min, followed by stimulation with NG or HG for additional 90 min. Intraplatelet ROS levels (E,H) , and phosphorylation of p38α MAPK (F,I) and p53 (G,J) were determined. Data were assessed by a one-way ANOVA followed by Dunnett’s t -test in (A–D) and by Tukey’s multiple comparisons test in (E–J) ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the vehicle control; # p < 0.05 and ## p < 0.01; ns, not significant difference.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Incubation, Control, Flow Cytometry, Phospho-proteomics, Western Blot

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Sulforaphane attenuates ROS/p53 signaling through downregulating AR-mediated activation of Src family kinases in HG-stimulated human platelets. (A) Washed human platelets were pre-incubated with SFN (5, 10, or 20 μM) or vehicle control for 40 min, followed by stimulation with NG or HG for additional 90 min. Platelets were lysed and Src phosphorylation was determined by Western blotting. (B) Washed human platelets were pre-incubated with SFN (20 μM) with or without ARI (10 μM) for 40 min, followed by stimulation with NG or HG for additional 90 min. Src phosphorylation was determined. (C–G) Washed human platelets were pre-incubated with SFN (20 μM) with or without a Src inhibitor PP2 (20 μM) for 40 min, followed by stimulation with NG or HG for additional 90 min. Src phosphorylation (C) , intraplatelet ROS levels (D,E) , and phosphorylation of p38α MAPK (F) and p53 (G) were determined. Data were assessed by a one-way ANOVA followed by Dunnett’s t -test in (A) and by Tukey’s multiple comparisons test in (B–G) ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the vehicle control; ns, not significant difference.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Activation Assay, Incubation, Control, Phospho-proteomics, Western Blot

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Sulforaphane attenuates AR-mediated platelet dysfunction mainly through downregulating Src/ROS/p53 signaling in HG-stimulated human platelets. Washed human platelets were pre-incubated with SFN (20 μM) with or without PP2 (20 μM) (A–D) , NAC (500 μM) (E–H) , or a p53 inhibitor PFT-μ (20 μM) (I–L) for 40 min, followed by stimulation with NG or HG for additional 90 min. Platelet Δψm dissipation (A,E,I) and PS exposure (B,F,J) were determined using flow cytometry. Platelet aggregation was stimulated by 1 μg/mL collagen (C,G,K) . Platelet surface expression of CD62P was analyzed by flow cytometry (D,H,L) . Data were assessed by a one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 3). * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the vehicle control; # p < 0.05, ## p < 0.01, and ### p < 0.001; ns, not significant difference.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Incubation, Flow Cytometry, Expressing, Control

Journal: Frontiers in Nutrition

Article Title: Sulforaphane attenuates aldose reductase-mediated platelet dysfunction in high glucose-stimulated human platelets via downregulation of the Src/ROS/p53 signaling pathway

doi: 10.3389/fnut.2025.1663245

Figure Lengend Snippet: Proposed mechanism of SFN in attenuating platelet dysfunction under HG conditions. SFN alleviates HG-induced platelet mitochondrial dysfunction, apoptosis, and hyperreactivity primarily through suppression of AR activity. This inhibition downregulates the Src/ROS/p53 signaling pathway, ultimately protecting against hyperglycemia-associated atherothrombosis by normalizing platelet function.

Article Snippet: Antibody against phospho-p53 was purchased from OriGene (MD, USA).

Techniques: Activity Assay, Inhibition