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: 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: Cancer Research

Article Title: Prophylactic In Vivo Hematopoietic Stem Cell Gene Therapy with an Immune Checkpoint Inhibitor Reverses Tumor Growth in Syngeneic Mouse Tumor Models

doi: 10.1158/0008-5472.can-19-1044

Figure Lengend Snippet: Figure 3. Effect of miR-423-5p target site overexpression on HSPCs. A, Vector structure. HDAd-GFP-miR-423 contains four miR-423-5p target sites in the 30UTR linked to the GFP gene. B, Mouse HSPCs (M; Lin cells from the bone marrow of CD46-transgenic mice) and human HSPCs (Hu; CD34þ cells) were infected with either HDAd-GFP or HDAd-GFP-miR423 at a multiplicity of infection of 500 or 3,000 vp/cell, respectively. Three days later, cell lysates were analyzed by Western blot analysis for CDKN1A. Blots were reprobed with anti-b-actin antibodies to adjust for loading differences. The right panel shows the quantification of CDKN1A signals normalized to b-actin signals. The signals from the corresponding mouse and human HDAd-GFP/mgmt samples were taken as 100%. C, Effect on progenitor colony formation. One day after HDAd infection, mouse Lin cells (2.5 103 cells per 35 mm dish) or human CD34þ cells (3 103 cells/dish) were plated for colony assays. Colonies were counted 12 days later (N ¼ 3; , P < 0.05). Statistical significance was calculated by two-sided Student t test (Microsoft Excel). [In agreement with previous studies (35, 36), infection of HSPCs at relatively high multiplicities of infection slightly reduced the colony forming capacity of HSPCs.] n.s., not significant.

Article Snippet: CDKN1A antibodies (STJ23069, St John's Laboratory) cross-reacted with the mouse and human protein. qRT-PCR for aPD-L1-g 1 mRNA qRT-PCR for aPD-L1-g1 mRNA is described in Supplementary Materials and Methods aPD-L1-g 1 ELISA Recombinant mouse PD-L1 protein (Sino Biological Inc., catalog no. 50010-M08H) at 2 mg/mL were used to coat ELISA plates.

Techniques: Over Expression, Plasmid Preparation, Transgenic Assay, Infection, Western Blot

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: IGF2 deficiency promotes liver aging through mitochondrial dysfunction and upregulated CEBPB signaling in D-galactose-induced aging mice.

doi: 10.1186/s10020-023-00752-0

Figure Lengend Snippet: Fig. 1 Liver tissues of old mice exhibit decreased expression of IGF2 and aging liver microenvironment. A Western blotting analysis for the protein expression of IGF2 and key senescence-associated genes P53, P21 and P16 in the liver from mice of different ages. The quantitative data are presented. mRNA levels of IGF2 (B), senescence-associated genes P53, P21 and P16 (C), and senescence-associated secretory phenotype genes IL-6, IL-1β, TNF-α and NF-κB1 (D), as determined by RT‒qPCR analysis. n = 5 per group. Data are shown as means ± SEM. One-way ANOVA was used for comparison among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Primary antibodies were diluted at 1:1000 and included IGF2 (ab9574, Abcam), P53 (10442-1- AP, Proteintech), P21 (A11454, Abclonal), P16 (A0262, Abclonal), CEBPB (sc-7962, Santa Cruz Biotechnology), AKT (9272S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), ERK1/2 (4695S, Cell Signaling Technology), p-ERK1/2 (4370S, Cell Signaling Technology), p38 (8690S, Cell Signaling Technology), p-p38 (4511S, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Comparison

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: IGF2 deficiency promotes liver aging through mitochondrial dysfunction and upregulated CEBPB signaling in D-galactose-induced aging mice.

doi: 10.1186/s10020-023-00752-0

Figure Lengend Snippet: Fig. 2 Conditional knockout of IGF2 in hepatocyte accelerated d-gal-induced liver aging. The expression levels of IGF2 were examined by Western blotting (A) and RT‒qPCR (B). Liver function evaluated by ALT (C) and AST (D). E Western blotting analysis for the protein level of senescence-associated genes P53, P21 and P16 in the liver tissues. The quantitative data are presented. F Relative mRNA expression levels of senescence-associated secretory phenotype genes IL-6, IL-1β, TNF-α and NF-κB1 in the liver tissues, as measured by RT‒qPCR. G SA-β-gal staining of liver sections. β-gal staining area analysis was shown. Scale bar, 100 um. H H&E staining of liver sections. Scale bar, 50 um. I Immunofluorescent staining of F4/80 and DAPI on liver sections. Fluorescence intensity analysis was shown. Scale bar, 50 um. A–F n = 5–7 per group, G–I n = 3 per group. All values are shown as means ± SEM. Dots represent individual level data. One-way ANOVA was used for comparison among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Primary antibodies were diluted at 1:1000 and included IGF2 (ab9574, Abcam), P53 (10442-1- AP, Proteintech), P21 (A11454, Abclonal), P16 (A0262, Abclonal), CEBPB (sc-7962, Santa Cruz Biotechnology), AKT (9272S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), ERK1/2 (4695S, Cell Signaling Technology), p-ERK1/2 (4370S, Cell Signaling Technology), p38 (8690S, Cell Signaling Technology), p-p38 (4511S, Cell Signaling Technology).

Techniques: Knock-Out, Expressing, Western Blot, Staining, Fluorescence, Comparison

Journal: Molecular medicine (Cambridge, Mass.)

Article Title: IGF2 deficiency promotes liver aging through mitochondrial dysfunction and upregulated CEBPB signaling in D-galactose-induced aging mice.

doi: 10.1186/s10020-023-00752-0

Figure Lengend Snippet: Fig. 6 Up-regulation of the CEBPB signaling is critical for the pro-aging effects of IGF2 deficiency. A Western blot analysis of protein levels of CEBPB in IGF2f/f and IGF2f/fCre mice of saline control group and d-gal model group. The quantitative data are presented. n = 5–7 per group. B Western blot analysis of protein levels of CEBPB in AML12 cells transfected with sh-IGF2 or sh-NC and treated with d-gal, H2O2 or the PBS control. C–H AML12 cells were transfected with sh-NC + si-NC, sh-NC + si-CEBPB, sh-IGF2 + si-NC and sh-IGF2 + si-CEBPB, and treated with d-gal or H2O2. C The senescence of AML12 cells detected by SA-β-Gal staining. β-gal staining area analysis was shown. Scale bar, 50 μm. D Immunofluorescence analysis of γ-H2AX expression in AML12 cells. Fluorescence intensity analysis was shown. Scale bar, 50 μm. Western blot analysis to measure the senescence-associated genes P53, P21 and P16 in AML12 cells treated with d-gal (E) or H2O2 (F). RT‒qPCR analysis of mRNA levels of senescence-associated secretory phenotype genes IL-6, IL-1β, TNF-α and NF-κB1 in AML12 cells treated with d-gal (G) or H2O2 (H). Data are shown as means ± SEM. One-way ANOVA was used for comparison among multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were repeated three times independently

Article Snippet: Primary antibodies were diluted at 1:1000 and included IGF2 (ab9574, Abcam), P53 (10442-1- AP, Proteintech), P21 (A11454, Abclonal), P16 (A0262, Abclonal), CEBPB (sc-7962, Santa Cruz Biotechnology), AKT (9272S, Cell Signaling Technology), p-AKT (4060S, Cell Signaling Technology), ERK1/2 (4695S, Cell Signaling Technology), p-ERK1/2 (4370S, Cell Signaling Technology), p38 (8690S, Cell Signaling Technology), p-p38 (4511S, Cell Signaling Technology).

Techniques: Western Blot, Saline, Control, Transfection, Staining, Immunofluorescence, Expressing, Fluorescence, Comparison

Journal: bioRxiv

Article Title: IRES-mediated translation of Δ160p53 regulates p53 functions and fine-tunes cancer homeostasis

doi: 10.64898/2026.08.21.744132

Figure Lengend Snippet: (A) Luciferase assay of H1299 cells after 48 h of transfection with pcDNA, FL-p53, and Δ160p53, and luciferase constructs (PG13 and pRL-TK); the PG13 construct is a p53 binding site-containing firefly luciferase plasmid, and pRL-TK is used as a transfection control. (B) Real-time PCR analysis of p21 mRNA levels in H1299 cells transfected with pcDNA, FL-p53, and Δ160p53 and luciferase constructs (PG13 and pRL-TK). (C) Western blot validation of FL-p53, Δ160p53, and p21 expression from H1299 cells transfected with pcDNA, FL-p53, and Δ160p53 and luciferase constructs (PG13 and pRL-TK), probed with p21 antibody, p53 polyclonal antibody, and β-actin antibody. (D) Luciferase assay of A549 cells transfected with pcDNA, Δ160p53, and luciferase constructs (PG13 and pRL-TK). (E) Luciferase assay of HCT116+/+ cells transfected with pcDNA, Δ160p53, and luciferase constructs (PG13 and pRL-TK). (F) Luciferase assay of HeLa cells after 48 h of transfection with pcDNA, Δ160p53, and luciferase constructs (PG13 and pRL-TK). (G) Western blot validation of FL-p53 and Δ160p53 from A549 cells transfected with pcDNA, Δ160p53, and luciferase constructs (PG13 and pRL-TK), probed with p53 polyclonal antibody and β-actin antibody. (H) Western blot analysis of FL-p53 and Δ160p53 from HCT116+/+ cells transfected with pcDNA, Δ160p53, and luciferase constructs (PG13 and pRL-TK), probed with p53 polyclonal antibody and β-actin antibody. (I) Western blot validation of FL-p53 and Δ160p53 in HeLa after 48 h of transfection with pcDNA, Δ160p53, and luciferase constructs (PG13 and pRL-TK), probed with p53 polyclonal antibody and β-actin antibody. Error bars indicate standard deviation (SD). All experiments were performed in three biological replicates (n = 3). A two-tailed Student’s t-test determined the criterion for significance (*P ≤ 0.05 or **P ≤ 0.01 or ***P ≤ 0.001).

Article Snippet: The BiP antibody (Abclonal, Cat. No. A4908), rabbit anti-GFP-Tag antibody (Abclonal, Cat. No. AE011), and p21 antibody (Abclonal, Cat. No. A19094) were used to probe Bip, GFP, and p21, respectively.

Techniques: Luciferase, Transfection, Construct, Binding Assay, Plasmid Preparation, Control, Real-time Polymerase Chain Reaction, Western Blot, Biomarker Discovery, Expressing, Standard Deviation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: TRAIL-receptor 2—a novel negative regulator of p53

doi: 10.1038/s41419-021-04048-1

Figure Lengend Snippet: A Whole-cell lysates of A549 wild type (WT) and TRAIL-R2-Sup (TR2 Sup) cells were analyzed by western blotting for the protein levels of TRAIL-R2, p53, MDM2, BAX, and p21. The level of β-Actin was determined in parallel and served as loading control. B mRNA levels of p21, MDM2 and BAX were analyzed by qRT-PCR in A549 cells and normalized to TBP. Bar chart shows mean values ± SD of three biological replicates ( n = 3). C Cell cycle analysis through PI staining followed by flow cytometry of A549 WT and TRAIL-R2-Sup cells. Bar chart shows mean values ± SD of three biological replicates ( n = 3). D Chromatin Immunoprecipitation (ChIP) was performed with anti-p53 (DO-1) and isotype control antibodies (IgG 2a ) on chromatin isolated from A549 WT and TRAIL-R2 Sup cells. DNA was extracted, and qRT-PCRs were performed using primers detecting the CDKN1A promotor. Enrichment was calculated as the fold increase in specific signal relative to the background signal. Results are shown ± SEM of four biological replicates ( n = 4). E A549 WT and TRAIL-R2 Sup cells were transiently transfected with expression vector coding for the long (TR2-long) or short (TR2-short) isoform of TRAIL-R2, each carrying a point mutation in the death domain, or with an empty vector (pCR3.1). After 48 h, protein levels of TRAIL-R2, p53, and p21 were analyzed by western blotting. The level of β-Actin was determined in parallel and served as loading control. Bands were analyzed by densitometry. Intensity of each band was normalized to the corresponding β-Actin. F A549 cells were treated with zVAD-fmk (20 µM) for 48 h. Whole-cell lysates were analyzed by western blotting for the expression of TRAIL-R2, p53, and p21. The level of β-Actin was determined in parallel and served as loading control. G A549 WT and TRAIL-R2 Sup cells were irradiated with 10 J/m 2 of UV-C radiation. After 16 h cell cycle analysis through PI staining followed by flow cytometry was performed. Bar chart shows mean values ± SD of three biological replicates ( n = 3). H A549 WT and TRAIL-R2 Sup cells were irradiated with 10 J/m 2 of UV-C radiation. After 6 h and 16 h whole-cell lysates were prepared and analyzed by western blotting for the expression of TRAIL-R2, p53, and p21. β-Actin was analyzed in parallel as loading control. * p < 0.05.

Article Snippet: The amount of extracted DNA was quantified via qPCR using Maxima SYBR Green/ROX master mix and a 7900HT Fast RT-PCR system (both Thermo Fisher Scientific) according to the manufacturer’s instructions. qPCR was performed with primers that detect the CDKN1A promotor [(6449), Cell Signaling Technology].

Techniques: Western Blot, Control, Quantitative RT-PCR, Cell Cycle Assay, Staining, Flow Cytometry, Chromatin Immunoprecipitation, Isolation, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Irradiation