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: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet: Muscle fibre cross-sectional area in oxidative ( A ) and non-oxidative ( B ) fibres from irradiated mice at 16 months of age. Frequencies of p21-positive myonuclei in oxidative ( C ) and glycolytic ( D ) fibres. ( E ) Telomere-associated foci (TAF) frequencies in myonuclei. Data are from at least five animals/group.

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques: Irradiation

Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet: Immunostaining and blotting methods.

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques: Immunostaining, Plasmid Preparation, Avidin-Biotin Assay, Immunohistochemistry-IF

Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet: List of metal conjugated antibodies for stress response pathway analysis by CyTOF.

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques:

Journal: eLife

Article Title: Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

doi: 10.7554/eLife.75492

Figure Lengend Snippet:

Article Snippet: p21 Waf1/Cip1 , 159Tb , 3159026 A , Fluidigm.

Techniques: Recombinant, 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 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

Journal: NPJ Regenerative Medicine

Article Title: Novel copper-ion coordinated andrographolide-loaded hydrogel activates Rac1/JNK1 axis for enhancing diabetic wound healing

doi: 10.1038/s41536-026-00457-y

Figure Lengend Snippet: A Venny plots of Andro target genes vs DWs causative genes. B PPI network highlighting key signaling proteins modulated by Andro treatment. Nodes represent individual proteins (colored by pathway/function; see legend); edges represent validated interactions. Major pathway clusters are annotated (e.g., JNK1/MAPK, Rac1/cell migration). C GO functional enrichment analysis of intersecting targets, showing significantly enriched biological processes (e.g., inflammatory response, cell migration, angiogenesis) ranked by gene count or significance. D KEGG pathway enrichment analysis ranked by gene count and significance. E Top 20 KEGG pathway enrichment analysis (sorted by enrichment score). F Representative docking pose of Andro within the proteins binding pocket, showing key noncovalent interactions.

Article Snippet: Rac1 protein (MCE, China) was diluted to 50 μg/mL in sodium acetate and immobilized onto the chip’s second channel at a flow rate of 10 μL/min.

Techniques: Migration, Functional Assay, Binding Assay

Journal: NPJ Regenerative Medicine

Article Title: Novel copper-ion coordinated andrographolide-loaded hydrogel activates Rac1/JNK1 axis for enhancing diabetic wound healing

doi: 10.1038/s41536-026-00457-y

Figure Lengend Snippet: A RMSD of complex, protein, and Ligand, reflecting structural deviation and conformational stability over time. B Rg of the complex, indicating overall compactness and structural tightness. C Rac1 and Andro binding site distance, monitoring binding stability and positional consistency. D FEL showing a single dominant basin, indicating conformational stability without large-scale reorganization. E Buried area between Andro and Rac1, representing the extent of buried surface and interaction strength. F Simulated conformational superposition, illustrating structural flexibility and binding site variations. G Modeling trajectory change, displaying dynamic shifts in binding site and molecular orientation. H PCA and protein structure, highlighting dominant motion modes and conformational flexibility. I Andro and Rac1 binding energies VDW and ELE, showing contributions from van der Waals and electrostatic interactions. J Hydrogen bond number, reflecting the stability and strength of polar interactions. K Hydrogen bond frequency between Andro and Rac1, indicating persistence and key residues involved in H-bond formation.

Article Snippet: Rac1 protein (MCE, China) was diluted to 50 μg/mL in sodium acetate and immobilized onto the chip’s second channel at a flow rate of 10 μL/min.

Techniques: Binding Assay

Journal: NPJ Regenerative Medicine

Article Title: Novel copper-ion coordinated andrographolide-loaded hydrogel activates Rac1/JNK1 axis for enhancing diabetic wound healing

doi: 10.1038/s41536-026-00457-y

Figure Lengend Snippet: A , B Representative western blots ( A ) and quantification ( B ) of Rac1, JNK1, Jun, and Fos protein levels in wound tissues at day 3 for each treatment group. C Relative mRNA expression levels of Rac1 , Jnk1 , Jun , and Fos in day-3 DW tissues. D SPR analysis confirming direct binding of Andro to Rac1 with a dissociation constant (KD) of 3.82 µM. E Relative mRNA expression levels of Rac1 , Jnk1 , Jun , and Fos in day-7 wound tissues. F , G Representative western blots ( F ) and quantification ( G ) of Rac1, JNK1, Jun, and Fos protein levels in day-7 wound tissues. The bar charts present mean ± SD ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs Control group, # P < 0.05, ## P < 0.01, ### P < 0.001, vs Matrix group. H Schematic model summarizing the proposed mechanism by which ASFH-delivered Andro treats DWs. Andro directly binds Rac1, activates the Rac1/JNK1/Jun/Fos signaling axis, promotes M1-to-M2 macrophage polarization, modulates cytokine profiles, enhances angiogenesis and collagen remodeling, and collectively accelerates wound closure.

Article Snippet: Rac1 protein (MCE, China) was diluted to 50 μg/mL in sodium acetate and immobilized onto the chip’s second channel at a flow rate of 10 μL/min.

Techniques: Western Blot, Expressing, Binding Assay, Control