Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a IB analysis of WCLs derived from HEK293T cells transfected with His-E4F1 and increasing doses of Flag-PA28γ. b Quantitative real-time PCR analysis of the indicated genes in HEK293T cells transfected with EV and increasing doses of Flag-PA28γ. Endo., endogenous. c HSC-3 cells stably expressing EV or Flag-PA28γ were treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of E4F1 levels relative to tubulin levels is shown. d , e HEK293T cells stably expressing EV or Flag-PA28γ were transfected with Myc-E4F1 truncations, and treated with 100 μgml −1 CHX for the indicated time before harvesting. Quantification of Myc-E4F1 levels relative to tubulin levels is shown. f HEK293T cells transfected with the indicated plasmids were treated without or with 10 μM MG132 for 6 h before harvesting, and WCLs were collected for IB analysis. g HEK293T cells stably expressing shRNA against endogenous PA28γ were transfected with the indicated plasmids, and WCLs were collected for IB analysis. h Purified PA28γ, E4F1 and 20S proteasome in the absence or presence of 100 nM proteasome inhibitor epoxomicin (Epox) were incubated as indicated for 45 min, followed by IB analysis. i Purified PA28γ WT, PA28γ-T23A, E4F1, p21 (as a positive control for E4F1) and 20S proteasome were incubated as indicated for 45 min, followed by IB analysis. Data in ( b – e ) represent the mean ± SD of three biological replicates; statistical significance was assessed by two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: Recombinant human E4F1 protein (H00001877-P01) and recombinant human p21/CDKN1A protein (NBP2-22976) were purchased from Novus Biologicals.

Techniques: Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, shRNA, Purification, Incubation, Positive Control

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Immunohistochemistry with primary anti-p21 antibody with positive reaction visible as brown staining. A – weak nuclear reaction in a grade 1 mast cell tumour (MCT); B – moderate nuclear reaction in a grade 2 MCT; C – strong nuclear reaction in a grade 3 MCT

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques: Immunohistochemistry, Staining

Journal: Journal of Veterinary Research

Article Title: Immunoreactivity of p21, MMP-1 and CB2 receptor proteins in cutaneous canine mast cell tumours: an association with the three-tier grading system

doi: 10.2478/jvetres-2023-0066

Figure Lengend Snippet: Frequency of immunoreactivity for p21 protein in grade 1, 2 and 3 mast cell tumours (MCTs). * – P-value < 0.05; **_ P-value < 0.01; *** P-value < 0.001 compared to grade 1 MCTs; (−) – no reaction; (+) – weak reaction; (++) – moderate reaction; (+++) – strong reaction

Article Snippet: The primary antibodies used in this study, all canine-specific, were as follows: anti-CB2R (Cat. No. TA317640; OriGene Technologies, Rockville, MD, USA), anti-p21 (Cat. No. M7202; clone SX118, Agilent Dako, Santa Clara, CA, USA) and anti-MMP-1 (Cat. No. ABIN2777120; antibodies-online, Aachen, Germany), diluted 1 : 200, 1 : 50 and 1 : 500, respectively.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Hydrogen Sulfide Inhibits High Glucose-Induced Neuronal Senescence by Improving Autophagic Flux via Up-regulation of SIRT1

doi: 10.3389/fnmol.2019.00194

Figure Lengend Snippet: Effect of hydrogen sulfide (H 2 S) on high glucose (HG)-elicited cellular senescence in HT22 cells. (A) Cell growth curves were generated using Trypan blue stain assays. (B) Representative images of senescent cells that were stained using senescence associated-β-galactosidase (SA-β-gal; Left, Magnification ×100) and quantitative analysis of the SA-β-gal positive cells (Right). The black arrows indicate the senescent cells, (C,D) The expressions of p16 INK4a and p21 CIP1 in HT22 cells were detected by Western blotting. Values were expressed as the mean ± SEM, n = 3. * P < 0.05, *** P < 0.001, vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. HG-treated alone group.

Article Snippet: Specific antibodies of p16 INK4a and p21 CIP1 were purchased from OriGene Technologies.

Techniques: Generated, Staining, Western Blot, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: Hydrogen Sulfide Inhibits High Glucose-Induced Neuronal Senescence by Improving Autophagic Flux via Up-regulation of SIRT1

doi: 10.3389/fnmol.2019.00194

Figure Lengend Snippet: Effects of CQ and 3-MA on the protection of H 2 S against HG-induced cellular senescence in HT22 cells. (A,E) Cell growth curves were generated by using Trypan blue stain assays. (B,F) Representative images of senescent cells that were stained for SA-β-gal (Left, Magnification ×10) and quantitative analysis of the SA-β-gal positive cells (Right). The black arrows indicate the senescent cells, n = 5. (C,D,G,H) The expressions of p16 INK4a and p21 CIP1 in HT22 cells were detected by Western blotting. Values were expressed as the mean ± SEM, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, vs. control group; # P < 0.05, ## P < 0.01, ### P < 0.001 vs. HG-treated alone group. & P < 0.05, && P < 0.01, &&& P < 0.001, vs. co-treated with NaHS and HG group.

Article Snippet: Specific antibodies of p16 INK4a and p21 CIP1 were purchased from OriGene Technologies.

Techniques: Generated, Staining, Western Blot, Control

Journal: International journal of oncology

Article Title: Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells.

doi: 10.3892/ijo_00000185

Figure Lengend Snippet: Figure 3. Aspirin induces p53 DNA binding activity as well as p21 protein and mRNA levels. A, shows time course of p53 DNA binding activity with aspirin treatment. Nuclear extracts were prepared from untreated and aspirin-treated (100 μM, 24 h) cells for the indicated times, incubated with

Article Snippet: Antiß-actin antibody and anti-Bax antibodies were obtained from Cell Signaling Technology Inc. Anti-acetyl p53 antibody was purchased from Cell Signaling Technology Inc. or Biolegend Inc. Anti-p21 antibody was purchased from Santa Cruz Biotechnology; anti-p53 monoclonal antibody was obtained from Zymed Inc; goat anti-rabbit IgG and goat anti-mouse IgG were from Bio-Rad; protease inhibitor cocktail tablets were obtained from Roche; p21 full length cDNA was obtained from Origene Inc.; all other chemicals were either from Sigma or Fisher.

Techniques: Binding Assay, Activity Assay, Incubation

Journal: International journal of oncology

Article Title: Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells.

doi: 10.3892/ijo_00000185

Figure Lengend Snippet: Figure 5. Aspirin inhibits CPT-induced p21 expression. For A, cells were left untreated or treated with aspirin alone (100 μM), CPT alone (100 nM) or aspirin and CPT for 24 h, cell lysates prepared and immunoblotted with anti-p21 antibody. B, the blot in A was stripped and reprobed with anti- acetyl-p53 antibody. C, the samples of A were immunoblotted with anti-p53 antibody. D, the samples of A were immunoblotted with anti ß-actin anti- body. For E, cells were left untreated or treated with aspirin alone (2.5 mM), CPT alone (100 nM) or aspirin and CPT for 24 h, cell lysates prepared and immunoblotted with anti-p21 antibody. F, the blot in E was stripped and reprobed with anti-ß-actin antibody. These experiments were repeated 3 times.

Article Snippet: Antiß-actin antibody and anti-Bax antibodies were obtained from Cell Signaling Technology Inc. Anti-acetyl p53 antibody was purchased from Cell Signaling Technology Inc. or Biolegend Inc. Anti-p21 antibody was purchased from Santa Cruz Biotechnology; anti-p53 monoclonal antibody was obtained from Zymed Inc; goat anti-rabbit IgG and goat anti-mouse IgG were from Bio-Rad; protease inhibitor cocktail tablets were obtained from Roche; p21 full length cDNA was obtained from Origene Inc.; all other chemicals were either from Sigma or Fisher.

Techniques: Expressing

Journal: International journal of oncology

Article Title: Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells.

doi: 10.3892/ijo_00000185

Figure Lengend Snippet: Figure 4. The anticancer drug CPT induces p21 in a sustained fashion. For A, cells were left untreated or treated with CPT (100 nM) for indicated time points, lysates prepared and immunoblotted with anti-p21 antibody. B, the blot in A was stripped and reprobed with anti-ß-actin antibody. C, the p21 bands in Figs. 3B and 4A were quantified using densitometric scanning and graphically represented. These experiments were repeated 4 times.

Article Snippet: Antiß-actin antibody and anti-Bax antibodies were obtained from Cell Signaling Technology Inc. Anti-acetyl p53 antibody was purchased from Cell Signaling Technology Inc. or Biolegend Inc. Anti-p21 antibody was purchased from Santa Cruz Biotechnology; anti-p53 monoclonal antibody was obtained from Zymed Inc; goat anti-rabbit IgG and goat anti-mouse IgG were from Bio-Rad; protease inhibitor cocktail tablets were obtained from Roche; p21 full length cDNA was obtained from Origene Inc.; all other chemicals were either from Sigma or Fisher.

Techniques:

Journal: International journal of oncology

Article Title: Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells.

doi: 10.3892/ijo_00000185

Figure Lengend Snippet: Figure 6. Aspirin does not inhibit CPT-induced p53 DNA binding activity. A, electrophoretic mobility shift assay. Cells were left untreated or treated with aspirin alone or CPT alone or co-treated with aspirin and CPT for 24 h, nuclear extracts prepared, and subjected to electrophoretic mobility shift assay using 32P-labeled p53 DNA binding element. For B, equal amount of nuclear extracts used in A were immunoblotted with anti-p21 antibody. For C, the blot in B was stripped and reprobed with anti-lamin B antibody. These experiments were repeated 3 times.

Article Snippet: Antiß-actin antibody and anti-Bax antibodies were obtained from Cell Signaling Technology Inc. Anti-acetyl p53 antibody was purchased from Cell Signaling Technology Inc. or Biolegend Inc. Anti-p21 antibody was purchased from Santa Cruz Biotechnology; anti-p53 monoclonal antibody was obtained from Zymed Inc; goat anti-rabbit IgG and goat anti-mouse IgG were from Bio-Rad; protease inhibitor cocktail tablets were obtained from Roche; p21 full length cDNA was obtained from Origene Inc.; all other chemicals were either from Sigma or Fisher.

Techniques: Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling

Journal: International journal of oncology

Article Title: Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells.

doi: 10.3892/ijo_00000185

Figure Lengend Snippet: Figure 7. Aspirin does not inhibit CPT-induced p21 mRNA levels. For A, cells were left untreated or treated with aspirin alone or CPT alone or co-treated with aspirin and CPT for 24 h, RNA isolated and subjected to Northern blot analysis as indicated in Materials and methods. B, shows the ethidium bromide-stained ribosomal 18S RNA pattern of the gel. These experiments were repeated 3 times.

Article Snippet: Antiß-actin antibody and anti-Bax antibodies were obtained from Cell Signaling Technology Inc. Anti-acetyl p53 antibody was purchased from Cell Signaling Technology Inc. or Biolegend Inc. Anti-p21 antibody was purchased from Santa Cruz Biotechnology; anti-p53 monoclonal antibody was obtained from Zymed Inc; goat anti-rabbit IgG and goat anti-mouse IgG were from Bio-Rad; protease inhibitor cocktail tablets were obtained from Roche; p21 full length cDNA was obtained from Origene Inc.; all other chemicals were either from Sigma or Fisher.

Techniques: Isolation, Northern Blot, Staining

Journal: International journal of oncology

Article Title: Aspirin inhibits camptothecin-induced p21CIP1 levels and potentiates apoptosis in human breast cancer cells.

doi: 10.3892/ijo_00000185

Figure Lengend Snippet: Figure 8. Lysate from aspirin-treated cells degrades recombinant GST-p21. For A, cytoplasmic extracts (prepared without protease inhibitors) from untreated cells were incubated with GST-p21. Separately, cytoplasmic extracts (prepared without protease inhibitors) from aspirin-treated (100 μM, 48 h) cells were incubated with GST-p21 recombinant protein in the presence or absence of protease inhibitor (PI) cocktail (Roche). After 4 h, the samples were immunoblotted using anti-p21 antibody. B, the blot in A was stripped and reprobed with anti-ß-actin antibody. These experiments were repeated 4 times. GST, glutathione-S transferase.

Article Snippet: Antiß-actin antibody and anti-Bax antibodies were obtained from Cell Signaling Technology Inc. Anti-acetyl p53 antibody was purchased from Cell Signaling Technology Inc. or Biolegend Inc. Anti-p21 antibody was purchased from Santa Cruz Biotechnology; anti-p53 monoclonal antibody was obtained from Zymed Inc; goat anti-rabbit IgG and goat anti-mouse IgG were from Bio-Rad; protease inhibitor cocktail tablets were obtained from Roche; p21 full length cDNA was obtained from Origene Inc.; all other chemicals were either from Sigma or Fisher.

Techniques: Recombinant, Incubation, Protease Inhibitor

Journal: Cell Cycle

Article Title: A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

doi: 10.4161/cc.23515

Figure Lengend Snippet: Figure 1. Identification of senescence-inducing kinase siRNAs. ( A ) Schematic overview of the high-content senescence screen. ( B ) Scatterplot analysis of data from the kinome HCS screen in p53+ cells. Cells were stained for Ki67, p53, p21 CIP1 and Hoechst at day 3, and multiparametric analysis was performed to determine siRNAs displaying decreased proliferation (< 0.5 StDev Ki67%) and an increased nuclear size (> 2.0 StDev Hoechst mean area). High, medium and low cell number indicators are assigned as the third parameter. ( C ) Senescence scores per gene are the sum of the senescence scores of its siRNA pool and the four individual siRNAs, omitting siRNAs conferring less than 50% specific gene knockdown. Scoring details are depicted in . References to studies establishing bona fide tumor suppressor functions for human ( Hs ) or mouse ( Mm ) genes are indicated.

Article Snippet: Primary antibodies were rabbit anti-p53 (Cell Signaling 928; 1:1500), goat anti-p21 (R&D Systems AF1047; 1:20.000) and mouse-anti-Ki67 (BD PharMingen 55600; 1:1500).

Techniques: Staining, Knockdown

Journal: Cell Cycle

Article Title: A high-content cellular senescence screen identifies candidate tumor suppressors, including EPHA3

doi: 10.4161/cc.23515

Figure Lengend Snippet: Figure 2. Delineation of senescence signatures. ( A ) hTERT-RPE1 p53 shRNA cells were transfected with kinome siRNAs in the absence of doxycycline and immunostained for p53 and p21 CIP1 following 72 h culture. Percentages of cells expressing p53, p21 CIP1 above threshold levels were calculated, and averages from four 384 wells are shown. ( B ) hTERT-RPE1 p53 shRNA cells were transfected with kinome siRNAs in the absence or presence of doxycycline and immunostained for Ki67. Percentages of cells expressing Ki67 protein above threshold levels were calculated, and averages from four 384 wells are shown. * indicates p ≤ 0,05 and ** indicates p ≤ 0,01. ( C ) Senescence scores per gene are the sum of the senescence scores of the siRNA pool plus four individual siRNAs, omitting siRNAs conferring less than 50% knockdown. Detailed scoring information is depicted in . ( D ) Quantitative p16 INK4A mRNA expression analyses during senescence induction. hTERT-RPE1 p53 shRNA cells were transfected with pooled siRNAs in the absence of doxycycline. RNA expression was quantitated using TaqMan analyses after 3 days of transfection. ( E ) Schematic model summarizing kinome screen data, using data depicted in ( A ) and ( D ). In an incipient tumor, modeled by hTERT-RPE1 cells, loss of selected tumor suppressors activates p53- and/or p16 INK4A -dependent senescence, and overt DNA damage. In a premalignant tumor, senescence may serve as a cell-intrinsic tumor suppressor mechanism to subvert oncogenic transformation. Loss of p16 INK4A and/or p53 promotes malignancy.

Article Snippet: Primary antibodies were rabbit anti-p53 (Cell Signaling 928; 1:1500), goat anti-p21 (R&D Systems AF1047; 1:20.000) and mouse-anti-Ki67 (BD PharMingen 55600; 1:1500).

Techniques: shRNA, Transfection, Expressing, Knockdown, RNA Expression, Transformation Assay