Journal: Aging (Albany NY)

Article Title: The protective effects of Phoenixin-20 in tumor necrosis factor α (TNF-α)-induced cell senescence of rheumatoid arthritis fibroblast-like synoviocytes (FLS)

doi: 10.18632/aging.205024

Figure Lengend Snippet: Phoenixin-20 downregulated p53 and p21 in TNF-α-treated RA-FLSs. RA-FLSs were stimulated with TNF-α (10 ng/mL) with or without Phoenixin-20 (10, 20 nM) for 7 days. ( A ) mRNA of p53 and p21; ( B ) Protein of p53 and p21 ( ※ P < 0.01 vs. vehicle group; # , ## , P < 0.05, 0.01 vs. TNF-α group, N = 5–6).

Article Snippet: The primary antibodies against p53 (#AB_2860544, 1:2000, Sino Biological, China), p21 (1:1000, #ab109520, Abcam, USA), STAT6 (#ab32108, 1:1500, Abcam, USA), and β-actin (1:10000, #ab6276, Abcam, USA) were imported.

Techniques:

Journal: Aging (Albany NY)

Article Title: The protective effects of Phoenixin-20 in tumor necrosis factor α (TNF-α)-induced cell senescence of rheumatoid arthritis fibroblast-like synoviocytes (FLS)

doi: 10.18632/aging.205024

Figure Lengend Snippet: Overexpression of STAT6 abolished the protective effect of Phoenixin-20 on TNF-α-induced cellular senescence in RA-FLSs. Cells were transduced with lentiviral STAT6, followed by stimulation with TNF-α (10 ng/mL) and Phoenixin-20 (20 nM) for 7 days. ( A ) Western blot analysis revealed successful overexpression of STAT6; ( B ) mRNA of p53 and p21. ( C ) Cellular senescence was assayed using SA-β-gal staining ( ※ P < 0.01 vs. vehicle group; ## P < 0.01 vs. TNF-α group, * P < 0.05 vs. PNX-20 group, N = 5–6).

Article Snippet: The primary antibodies against p53 (#AB_2860544, 1:2000, Sino Biological, China), p21 (1:1000, #ab109520, Abcam, USA), STAT6 (#ab32108, 1:1500, Abcam, USA), and β-actin (1:10000, #ab6276, Abcam, USA) were imported.

Techniques: Over Expression, Transduction, Western Blot, Staining

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: Molecular Cancer

Article Title: Differential susceptibility and role for senescence in CART cells based on costimulatory domains

doi: 10.1186/s12943-025-02371-1

Figure Lengend Snippet: Recurrent activation/rest differentially induced senescence in CART cells with alternative costimulatory domains. A-E. The percentage of CD3 + T cells that are positive for ( A. ) p21, ( B. ) KLRG1, ( C. ) uPAR, ( D. ) CD27 and ( E. ) TIM-3 as measured by flow cytometry are plotted ( n = 6 donors) (A-D., two-way ANOVA, E., t-test). F-G. Expression of uPAR, TIM-3, and KLRG1 and loss of CD27 expression are considered senescence events. Simultaneous occurrence of two, three, and four of these events in ( F. ) D8 and ( G. ) D15 CART cells are plotted as percent of CD3 + cells (t-test). H. DNA is isolated from D0, D8 and D15 CART cells. The telomere lengths are measured by qPCR and plotted for each time point (two-way ANOVA). I-L. mRNA expression of ( I. ) p21, ( J. ) p53, ( K. ) PIM-1, and ( L. ) uPAR are measured by qPCR. Fold change is calculated by 2ˆ (–delta delta CT). The expression levels are normalized to BBζ D8 (two-way ANOVA). M. Recurrently activated CART cells were immunoblotted for DNA damage markers p-H2AX (Ser139), p-ATM (Ser1981), and p-Chk2 (Thr68). N. NSG mice received JeKo-1, were randomized, and received BBζ and 28ζ. The spleens from mice were collected and flowed for the indicated markers 21 days after CART injection ( n = 3, t-test). Error bars, SEM. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: p21 Waf1/Cip1 (cat# CST 8865) and p53 (cat# CST 5429) antibodies were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA).

Techniques: Activation Assay, Flow Cytometry, Expressing, Isolation, Injection

Journal: Molecular Cancer

Article Title: Differential susceptibility and role for senescence in CART cells based on costimulatory domains

doi: 10.1186/s12943-025-02371-1

Figure Lengend Snippet: Recurrent activation/rest induces senescence through MYC activation in BBζ. A. MYC is overexpressed in CART cells through lentiviral transduction. MYC expression is measured by qPCR. Fold change is calculated by 2ˆ (–delta delta CT). The expression levels are normalized to control (Cont) for each CART cell type. B . DNA damage and senescence makers are measured by immunoblotting of the indicated CART groups (representative of two donors). C. p53 levels of indicated CART groups as measured by flow cytometry are shown. (t-test, error bars, SEM, three technical replicates). D-F. MYC OE CART cells were cocultured with JeKo-1 cells. Cycling cells were measured by ( D. ) EDU, ( E. ) cytotoxicity, and ( F. ) proliferation (t-test, each point represents the average of three technical replicates from individual biological donors). G. JeKo-1-bearing mice received control or wildtype MYC-overexpressing CART cells. Peripheral blood samples are collected from mice to measure the number of circulating CART cells (t-test). H-I. The tumor burdens measured by bioluminescence were assessed weekly (two-way ANOVA). * p < 0.05, ** p < 0.01, *** p < 0.001. J. Kaplan-Meier survival curve with MYC OE vs. Cont CART cells are shown (Log-rank test). Error bars, SEM. * p < 0.05, ** p < 0.01

Article Snippet: p21 Waf1/Cip1 (cat# CST 8865) and p53 (cat# CST 5429) antibodies were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA).

Techniques: Activation Assay, Transduction, Expressing, Control, Western Blot, Flow Cytometry