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: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: p21 in MDA‐MB‐231 cells treated with abemaciclib and/or ABT‐263. (A, B) Cancer cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 2 days. After harvesting, the cytoplasmic and nuclear fractions were separated and subjected to immunoblotting. TBP and GAPDH were used as controls. (C) Immunoblotting was performed similarly using whole lysates. (D) MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) with zVAD (10 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. The numbers are proportions of the subsets. (Left) Data of the means ± SD of three cells are shown. * p < 0.05, ** p < 0.01. (E) MDA‐MB‐231 cells were treated similarly with zVAD (10 µM) for 24 h and subjected to immunoblotting. (F) Untreated MDA‐MB‐231 cells were cultured with Hoechst 33342 (5 µg/ml) and stained with anti‐p21 and anti‐caspase‐3 antibodies followed by an Alexa 488‐conjugated anti‐rabbit antibody and Cy5‐conjugated anti‐mouse IgG. Confocal imaging reveals nuclei (blue), p21 (green), and caspse‐3 (white). Scale, 10 µm. (G) Control and p21‐overexpressing MDA‐MB‐231 cells were examined for their p21 expression by immunoblotting. (H) Control and p21‐overexpressing MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐APC. Numbers are the proportions of the subset (Left). The means ± SD of four wells are shown. ** p < 0.01. (I) Cancer cells were treated with abemaciclib (1.5 µM) for 24 h, and the cell lysates were subjected to immunoblot to examine the expression of p21 and c‐Myc

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Western Blot, Staining, Cell Culture, Imaging, Control, Expressing

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Effect of genetic knockdown of p21 in MCF‐7 cells on their sensitivity to drugs. (A) siRNA‐transfected cancer cells were cultured for 48 h and subjected to immunoblotting. (B) MDA‐MB‐231 and MCF‐7 cells transfected with siRNA p21(I) 2 days prior were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 48 h. After staining with annexin V‐FITC and PI, flow cytometric analysis was performed. Data are the means of three wells. * p < 0.05, ** p < 0.01. (C) Representative flow cytometry results; numbers are percentages of the subsets. (D) MDA‐MB‐231 cells were cultured with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) in the presence of caspase inhibitors (10 µM) for 48 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. Data are the means of three wells. ** p < 0.01. (E) siRNA p21(I)‐transfected MDA‐MB‐231 and MCF‐7 cells were cultured with TRAIL for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. ** p < 0.01

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Knockdown, Transfection, Cell Culture, Western Blot, Staining, Flow Cytometry

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Poor prognosis of breast cancer patients with p21 high compared with those with p21 low . (A, B, C) Kaplan–Meier plotter univariate analysis of survival time in CDKN1A mRNA expression in breast cancer. Version 2021 of the database was used for analysis. Outlier array data were excluded for array quality control. Patients were split into low‐ and high‐expression groups based on the optimal cutoff. (D, E) TRGAted was used for survival analysis according to p21 protein level in patients with invasive breast carcinoma. All subtypes of TCGA‐BRCA‐L4 were used in the analysis. Patients were split into low‐ and high‐expression groups based on the optimal cutoff

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Expressing, Control

Journal: Experimental cell research

Article Title: KIF20A promotes the development of fibrosarcoma via PI3K-Akt signaling pathway.

doi: 10.1016/j.yexcr.2022.113322

Figure Lengend Snippet: Fig. 2. KIF20A downregulation inhibits cell prolifer ation, migration and invasion in vitro. (A) After Kif20a gene knockdown, cell cycle was measured by flow cytometry. (B) KIF20A, Cyclins and cyclin-dependent kinase inhibitor p21Waf1/Cip1 levels were measured by Western blotting. GAPDH was used as an internal control. (C) Cell proliferation was evaluated by CCK-8 assay at 12, 24, 36, and 48 h. (D) Cell proliferation was evaluated by cell clone formation assay. (E) Cell migration was measured via Transwell assay. (F) Cell invasion was measured via Transwell assay, and matrigel was added to the upper chambers of Trans well. Scale bar represents 10 μm. The cells used were stably-transfected HT-1080. Data are representative of 3 independent experiments. *: p < 0.05, **: p < 0.01, ***p < 0.001.

Article Snippet: The primary antibody for KIF20A was purchased from Santa Cruz (USA); GAPDH, β-Actin, Phospho-Akt Pathway Antibody Sampler Kit #9916, NF-κB Pathway Sampler Kit #9936, PI3K, p21 Waf1/Cip1 (DCS60), Cyclin E1, Cyclin A2, Caspase 3, Caspase 7, Caspase 8, Caspase 9, Bax, and Bcl-2 were purchased from Cell Signaling Technology (USA).

Techniques: Migration, In Vitro, Knockdown, Flow Cytometry, Western Blot, Control, CCK-8 Assay, Tube Formation Assay, Transwell Assay, Stable Transfection, Transfection

Journal: Cancers

Article Title: TGF-β/SMAD Pathway Is Modulated by miR-26b-5p: Another Piece in the Puzzle of Chronic Lymphocytic Leukemia Progression

doi: 10.3390/cancers14071676

Figure Lengend Snippet: mRNA levels expression of SMAD proteins and of specific target genes modulated by TGF-β pathway activation. Relative expression of SMAD-2, SMAD-3, SMAD-4, SMAD-7, p21 −Cip1 , and KLF10 mRNAs were compared by q-PCR between progressive and stable U-CLL subgroups. Mann–Whitney Unpaired test and median with 95% CI are depicted. GAPDH was used as an endogenous control, and cDNA of MEC-1 cells were used as a reference sample. The relative expression was calculated as 2 −ΔΔCt . In all cases, p < 0.05 was considered statistically significant, (* = p < 0.05, ** = p < 0.01, and **** = p < 0.0001, ns = not significant).

Article Snippet: For intracellular detection, after surface membrane staining with anti-CD19-percpCy5.5 anti-CD5-fitc (1/50) (Biolegend), cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences, San Jose, CA, USA) and incubated with α-Smad-4-Alexa 647 (1/25) and p21 −Cip1 rabbit mAb (1/200) (Cell signaling) followed by goat antirabbit IgG-Cy5 (1/1000) (ThermoFisher, Waltham, MA, USA).

Techniques: Expressing, Activation Assay, MANN-WHITNEY, Control

Journal: Cancers

Article Title: TGF-β/SMAD Pathway Is Modulated by miR-26b-5p: Another Piece in the Puzzle of Chronic Lymphocytic Leukemia Progression

doi: 10.3390/cancers14071676

Figure Lengend Snippet: ( A – C ) Evaluation of mRNA and protein expression levels and cellular localization of SMAD-4 in CLL cells. ( A ) Correlation between miR-26b-5p and SMAD-4 mRNA expression in the U-CLL subgroups. Red circles = Progressive U-CLL, and green circles = Stable U-CLL ( p ≤ 0.0001, Spearman’s rank test, r = 0.81, n = 26). ( B ) SMAD-4 protein expression evaluated by flow cytometry in CLL cells (CD19+/CD5+) in the different U-CLL subgroups ( p = 0.013, Mann–Whitney Unpaired test, n = 19). ( C ) Confocal microscopy of Smad-4 (red) and IgM (green) from two representative patients; stable (CLL#10) and progressive (CLL#05) are depicted. Scale bar: 5 µm. Specific fluorescence intensity in nucleus and cytoplasm was measured in the different subgroups. Between 100 and 200 cells were counted in each sample ( n = 15; stable U-CLLs, n = 7; and progressive U-CLLs, n = 8). Next, MFI in the nucleus and cytoplasm was used to obtain integrated density and compare mean fluorescence intensities of Smad-4 in the nuclei of CLL samples, ( p = 0.040, Mann–Whitney Unpaired test, n = 15). ( D ) p21 −Cip1 protein expression evaluated by flow cytometry gating CD19+/CD5+ cells and c-MYC mRNA relative expression from stable and progressive U-CLLs ( p = 0.007, n = 17 and p = 0.004, n = 25, respectively, Mann–Whitney Unpaired test) are depicted. In all cases, p < 0.05 was considered statistically significant, (* = p < 0.05, ** = p < 0.01, and *** = p < 0.001, ns = not significant).

Article Snippet: For intracellular detection, after surface membrane staining with anti-CD19-percpCy5.5 anti-CD5-fitc (1/50) (Biolegend), cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences, San Jose, CA, USA) and incubated with α-Smad-4-Alexa 647 (1/25) and p21 −Cip1 rabbit mAb (1/200) (Cell signaling) followed by goat antirabbit IgG-Cy5 (1/1000) (ThermoFisher, Waltham, MA, USA).

Techniques: Expressing, Flow Cytometry, MANN-WHITNEY, Confocal Microscopy, Fluorescence

Journal: Cancers

Article Title: TGF-β/SMAD Pathway Is Modulated by miR-26b-5p: Another Piece in the Puzzle of Chronic Lymphocytic Leukemia Progression

doi: 10.3390/cancers14071676

Figure Lengend Snippet: Inhibition of miR-26b-5p and evaluation of SMAD-4, p21 −Cip1 , and c-MYC expression changes in primary CLL cells of progressive U-CLL patients. ( A ) Representative dot plots showing transfected CLL cells with specific antagomir targeting miR-26b-5p labeled with Dy547 (left panel). Graph shows relative expression levels of mir-26b-5p in CLL cells after transfection with the specific antagomir and the irrelevant miR-67 control ( p = 0.015, signed-rank test, n = 7). Agarose gel 5% stained with Ethidium Bromide shows a representative patient after transfection with the specific antagomir (Tant) and the corresponding controls (not transfected—NT—and transfected with the irrelevant miR-67, Tctrol) (right panel). ( B – D ) Percentages of cells expressing Smad-4 ( B ), p21 −Cip1 ( C ), and mRNA levels expression of c-MYC ( D ) after transfection with the specific inhibitor of miR-26b-5p and with miR-67 (control) in each patient ( p = 0.031, p = 0.015, and p = 0.047, respectively, Wilcoxon signed-rank test, n = 7). Each color corresponds to a single patient. In all cases, p < 0.05 was considered statistically significant, (* = p < 0.05).

Article Snippet: For intracellular detection, after surface membrane staining with anti-CD19-percpCy5.5 anti-CD5-fitc (1/50) (Biolegend), cells were fixed and permeabilized (Cytofix/Cytoperm, BD Biosciences, San Jose, CA, USA) and incubated with α-Smad-4-Alexa 647 (1/25) and p21 −Cip1 rabbit mAb (1/200) (Cell signaling) followed by goat antirabbit IgG-Cy5 (1/1000) (ThermoFisher, Waltham, MA, USA).

Techniques: Inhibition, Expressing, Transfection, Labeling, Control, Agarose Gel Electrophoresis, Staining

Journal: Antioxidants

Article Title: Epicatechin Gallate Ameliorates UVB-Induced Photoaging by Inhibiting p38α-Mediated Autophagy and Oxidative Stress

doi: 10.3390/antiox15020180

Figure Lengend Snippet: Effects of ECG on UVB-induced senescence markers in HaCaT cells. ( A , B ) Cell cycle distribution analysis of HaCaT cells by flow cytometry. The histogram in ( A ) shows DNA content (propidium iodide staining intensity) on the x-axis and cell count on the y-axis. The stacked bar chart in ( B ) quantifies the percentage of cells in different cell cycle phases (G0/G1, S, and G2/M). ( C ) Relative mRNA expression levels of p53 , p16 , p21 , IL6 , and TNF-α determined by RT-qPCR. ( D , E ) Representative Western blots and densitometric analysis of p53, p16, and p21 proteins. ( F , I ) Representative immunofluorescence images of γH2AX and Lamin B1 (scale bar: 50 μm). γH2AX foci are shown in green, Lamin B1 is shown in red, and nuclei are counterstained with DAPI (blue). White arrows indicate nuclei with damage. ( G ) Representative images of SA-β-gal staining in HaCaT cells (scale bar: 200 μm). ( H ) Quantitative analysis of SA-β-gal positive cells. Data are presented as the mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: Rabbit anti-Beclin1 (BECN1) antibody (cat. #11306), mouse anti-tubulin antibody (cat. #66031-1-IG), rabbit anti-LaminB1 (LMNB1) antibody (cat. #12987-1-AP), rabbit anti-Cyclin-Dependent Kinase Inhibitor 1A (p21, CDKN1A) antibody (cat. #10355-1-AP) and rabbit anti-LC3 antibody (cat. #14600) were purchased from Proteintech group (Wuhan, China).

Techniques: Flow Cytometry, Staining, Cell Characterization, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence

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: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: p21 in MDA‐MB‐231 cells treated with abemaciclib and/or ABT‐263. (A, B) Cancer cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 2 days. After harvesting, the cytoplasmic and nuclear fractions were separated and subjected to immunoblotting. TBP and GAPDH were used as controls. (C) Immunoblotting was performed similarly using whole lysates. (D) MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) with zVAD (10 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. The numbers are proportions of the subsets. (Left) Data of the means ± SD of three cells are shown. * p < 0.05, ** p < 0.01. (E) MDA‐MB‐231 cells were treated similarly with zVAD (10 µM) for 24 h and subjected to immunoblotting. (F) Untreated MDA‐MB‐231 cells were cultured with Hoechst 33342 (5 µg/ml) and stained with anti‐p21 and anti‐caspase‐3 antibodies followed by an Alexa 488‐conjugated anti‐rabbit antibody and Cy5‐conjugated anti‐mouse IgG. Confocal imaging reveals nuclei (blue), p21 (green), and caspse‐3 (white). Scale, 10 µm. (G) Control and p21‐overexpressing MDA‐MB‐231 cells were examined for their p21 expression by immunoblotting. (H) Control and p21‐overexpressing MDA‐MB‐231 cells were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 24 h. Flow cytometric analysis was done after staining with annexin V‐APC. Numbers are the proportions of the subset (Left). The means ± SD of four wells are shown. ** p < 0.01. (I) Cancer cells were treated with abemaciclib (1.5 µM) for 24 h, and the cell lysates were subjected to immunoblot to examine the expression of p21 and c‐Myc

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Western Blot, Staining, Cell Culture, Imaging, Control, Expressing

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Effect of genetic knockdown of p21 in MCF‐7 cells on their sensitivity to drugs. (A) siRNA‐transfected cancer cells were cultured for 48 h and subjected to immunoblotting. (B) MDA‐MB‐231 and MCF‐7 cells transfected with siRNA p21(I) 2 days prior were treated with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) for 48 h. After staining with annexin V‐FITC and PI, flow cytometric analysis was performed. Data are the means of three wells. * p < 0.05, ** p < 0.01. (C) Representative flow cytometry results; numbers are percentages of the subsets. (D) MDA‐MB‐231 cells were cultured with abemaciclib (1.5 µM) and ABT‐263 (1.5 µM) in the presence of caspase inhibitors (10 µM) for 48 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. Data are the means of three wells. ** p < 0.01. (E) siRNA p21(I)‐transfected MDA‐MB‐231 and MCF‐7 cells were cultured with TRAIL for 24 h. Flow cytometric analysis was done after staining with annexin V‐FITC and PI. ** p < 0.01

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Knockdown, Transfection, Cell Culture, Western Blot, Staining, Flow Cytometry

Journal: Cancer Medicine

Article Title: Protective role of cytoplasmic p21Cip1/Waf1 in apoptosis of CDK4/6 inhibitor‐induced senescence in breast cancer cells

doi: 10.1002/cam4.4410

Figure Lengend Snippet: Poor prognosis of breast cancer patients with p21 high compared with those with p21 low . (A, B, C) Kaplan–Meier plotter univariate analysis of survival time in CDKN1A mRNA expression in breast cancer. Version 2021 of the database was used for analysis. Outlier array data were excluded for array quality control. Patients were split into low‐ and high‐expression groups based on the optimal cutoff. (D, E) TRGAted was used for survival analysis according to p21 protein level in patients with invasive breast carcinoma. All subtypes of TCGA‐BRCA‐L4 were used in the analysis. Patients were split into low‐ and high‐expression groups based on the optimal cutoff

Article Snippet: The following siRNAs were used: p21(I) siRNA (#6456; CST), p21(II) siRNA (#6558; CST), and control siRNA (#6568; CST).

Techniques: Expressing, Control