Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in aged mouse plasma and myocardial tissue. (A) Relative expression levels of p16 and p21 in myocardial tissues of young mice (aged 2 months) and aged mice (aged 15 months) were detected by western blotting. (B) Relative mRNA expression levels of adiponectin, AdipoR1 and APPL1 in the plasma of young mice and aged mice were detected by reverse transcription-quantitative PCR. (C) Relative expression levels of adiponectin, AdipoR1 and APPL1 in myocardial tissues of young mice and aged mice were analyzed using western blot analysis. **P<0.01, ***P<0.001 vs. young mice. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Clinical Proteomics, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Expression of adiponectin, AdipoR1 and APPL1 is downregulated in D-gal-treated cardiomyocytes. (A) Relative expression levels of p16 and p21 in H9c2 cells treated with 2.5, 5 and 10 g/l of D-gal were analyzed using western blot analysis. *P<0.05, **P<0.01, ***P<0.001 vs. control. Transfection with Ov-adiponectin promotes the expression of AdipoR1 and APPL1 in D-gal-treated cardiomyocytes. (B) Relative mRNA expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed via RT-qPCR. ***P<0.001 vs. control; ### P<0.001 vs. D-gal + Ov-NC. (C) Relative protein expression of adiponectin in H9c2 cells before and after transfection with Ov-adiponectin plasmid was analyzed using RT-qPCR. ***P<0.001 vs. control; ## P<0.01 vs. D-gal + Ov-NC. (D) Relative mRNA expression levels of AdipoR1 and APPL1 in H9c2 cells before and after transfection with Ov-adiponectin plasmids were detected using RT-qPCR. ***P<0.001 vs. control; ### P<0.001 vs. D-gal + Ov-NC. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; D-gal, D-galactose; Ov-, overexpression.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Western Blot, Control, Transfection, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Over Expression

Journal: Molecular Medicine Reports

Article Title: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1

doi: 10.3892/mmr.2021.12358

Figure Lengend Snippet: Adiponectin inhibits D-gal-induced cardiomyocyte senescence via AdipoR1/APPL1. (A and B) Relative expression levels of AdipoR1 in D-gal-treated H9c2 cells with Ov-adiponectin before and after transfection with si-AdipoR1 and si-APPL were measured using RT-qPCR. **P<0.01, ***P<0.001 vs. D-gal + Ov-adiponectin. (C and D) Relative expression levels of APPL1 in D-gal-treated H9c2 cells with Ov-adiponectin before and after transfection with si-AdipoR1 and si-APPL were measured using RT-qPCR. ***P<0.001 vs. D-gal + Ov-adiponectin. (E) Cellular senescence in the different groups was detected using SA-β-gal staining. (F) Relative expression levels of p16 and p21 in the different groups were detected using western blot analysis. ***P<0.001 vs. control; ## P<0.01, ### P<0.001 vs. D-gal + Ov-NC; $ P<0.05 vs. D-gal + Ov-adiponectin + negative-NC. AdipoR1, adiponectin receptor 1; APPL1, adaptor protein phosphotyrosine interacting with PH domain and leucine zipper 1; Ov-, overexpression; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; si-, small interfering RNA; D-gal, D-galactose; SA-β-gal, senescence-associated β-galactose.

Article Snippet: The antibodies were as follows: Anti-p16 (1:1,000; cat. no. 18769; Cell Signaling Technology, Inc.), anti-p21 (1:1,000; cat. no. 2947; Cell Signaling Technology, Inc.), anti-adiponectin (1:1,000; cat. no. 2789; Cell Signaling Technology, Inc.), anti-AdipoR1 (1:800; cat. no. bs-0610R; BIOSS), anti-APPL1 (1:1,000; cat. no. 3858; Cell Signaling Technology, Inc.), anti-heme oxygenase (HO)-1 (1:1,000; cat. no. 43966; Cell Signaling Technology, Inc.), anti-high mobility group box 1 (HMGB1; 1:1,000; cat. no. 3935; Cell Signaling Technology, Inc.) and mouse anti-rabbit secondary antibody (1:1,000; cat. no. 5127; Cell Signaling Technology, Inc.).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Staining, Western Blot, Control, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control, Small Interfering RNA

Journal: BMC Biotechnology

Article Title: Modified gateway system for double shRNA expression and Cre/lox based gene expression

doi: 10.1186/1472-6750-11-24

Figure Lengend Snippet: Successful shRNA/cDNA targeting using designed entry vectors . A) pENTR constructs targeting exon 2 of p16 were transfected into 293T cells. RNA was isolated 48 hours post transfection and the mRNA expression level was determined using Q-RT-PCR. The average represents fold gene expression level relative to pENTRhU6hH1 transfected 293T cells (Students t-test; p < 0.01). B) pENTR constructs for CCND1 and/or CCND3 shRNA expression were recombined with plko.puro.DEST. Western blot shows the successful knockdown of CCND1/CCND3 in Panc1 cells. C) pENTR constructs for p16 shRNA expression were recombined with plko.YFP.DEST. Western blot shows p16 knockdown in H6c7 cells transfected with p16 shRNA constructs. D) pENTR.Cre mediated Cdk4 overexpression in 293T cells transiently transfected with pENTR.Cdk4.ON. The average represents fold gene expression level relative to control vector cell line f) Luciferase activity downregulation by Cre protein in stable HeLa cells overexpressing Luc2 mediated by plko.dsRed2.Luc2.ON lentivirus.

Article Snippet: Immunoblotting was performed using whole protein extracts and probed with the following antibodies against cyclin D3 (BD Biosciences, San Jose, CA), cyclin D1 (BD Biosciences), p16 INK4a (Cell Signalling, Boston, MA) and p14 ARF (NeoMarkers, Freemont, CA), GAPDH (Abcam, Cambridge, MA), and secondary mouse or rabbit conjugated IgG-horseradish peroxidase (Cell Signalling).

Techniques: shRNA, Construct, Transfection, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Western Blot, Knockdown, Over Expression, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Schematics of the experimental strategy to measure senescence markers (p21, p16 and SA-βGal activity) and SASP cytokines levels in mice lungs at 10 and 20 days p.i. Day 1 CFU= 55. (b) %p21+p16+ and (c) %SA-βGal+ (CellEvent Senescence green+) cells out of all live lung (uninfected and Mtb- infected mice) and Spleen ( Mtb- infected mice) cells at 10 days p.i. as determined by multicolor flow cytometry. (d) %p21+p16+ and (e) %SA-βGal+ cells out of different lung-cell types at 10 days p.i., as determined by multicolor flow cytometry. Gating for p16⁺, p21⁺, and SA-βGal⁺ events for each cell type were established using reference cells from Mtb -infected WT B6 mice (set at 1 %). (AMs: Alveolar macrophages, IMs: Interstitial macrophages) (f) Normalized concentration of SASP-cytokines in lung homogenates at 20 dpi and 4 wpi as measured by a LEGENDplex™ Mouse Inflammation Panel (13-plex). The data are means ± SEM. Each data point represents a mouse (n=11-12). (two-way ANOVA with Tukey’s (b-e) or Dunnett’s (f) multiple comparisons test).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Activity Assay, Infection, Flow Cytometry, Concentration Assay

Journal: bioRxiv

Article Title: Elimination of senescent cells with senolytic host-directed therapy reduces tuberculosis progression in mice

doi: 10.1101/2025.03.28.645957

Figure Lengend Snippet: (a) Representative H&E-stained images of Mtb -infected mice lungs treated with Vehicle or drugs, and respective ImageJ-based quantification. Yellow arrow indicates necrotic granuloma in Vehicle treated B6.Sst1S mice at 5 wpi. The data are means ± SEM. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. Square represents necrotic granuloma formation in the lung. (b) Representative γH2A.X-Immunohistochemistry images of mice after lungs vehicle/ drugs treatment, and respective Violin plots to show ImageJ quantification of γH2A.X-stained area. Each data point represents a mouse. Statistical analysis between two groups was done by unpaired two-tailed Student’s t test. (c) %p21+βGal+ and %p16+βGal+ subpopulation observed in all live lung cells of Mtb -infected mice (n= 5-8). The data are means ± SEM. Each data point represents a mouse. Statistical analysis was calculated by two-way ANOVA with Dunnett’s multiple comparisons test. Bubble plot to show %p21+βGal+ and %p16+βGal+ subpopulation in different lung cell types in Mtb -infected (d) B6.Sst1S mice (n= 8) and (e) WT B6 old mice (n= 5) at indicated time point and treatment groups. The size of the bubble indicates %subpopulation out of all of particular cell types and color is adjusted p values relative to Veh group. Statistics are calculated by one-way ANOVA with Tukey’s multiple comparisons test ( p > 0.15: ns).

Article Snippet: The PVDF membranes were blocked using 5% Blotting-grade blocker (Bio-Rad; 1706404) in TBST buffer-Tris-buffered saline (Quality Biological; 351086101)+ 0.1% Tween 20 (Sigma; P2287) for 1-2 hour and incubated overnight at 4°C with the primary antibodies from Mouse Reactive Senescence Marker Antibody Sampler Kit (Cell Signaling Technology-78551)-Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb (1: 1000 dilution), Lamin B1 (E6M5T) Rabbit mAb (1: 1000 dilution), HMGB1 (D3E5) Rabbit mAb (1: 1000 dilution), p16 INK4A (E5F3Y) Rabbit mAb (1: 1000 dilution) and β-Actin (D6A8) Rabbit mAb (Cell Signaling Technology; 8457) (1:5000 dilution).

Techniques: Staining, Infection, Two Tailed Test, Immunohistochemistry

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 1 Abnormally high expression of ARPC5 in glioma. (A) Differential expression of ARPC5 in various tumor types was analyzed based on the TIMER online website. (B) The distinct upregulation of ARPC5 in glioma was demonstrated in GEPIA online website. (C) Box plot based on the expression of ARPC5 in the GSE2223 (Glioma = 50, Normal = 4). (D) Box plot based on the expression of ARPC5 in the GSE29796 (Glioma = 52, Normal = 20). (E) Box plot based on the expression of ARPC5 in the GSE116520 (Glioma = 34, Normal = 8). (F) Box plot based on the protein expression of ARPC5 in the CPTAC samples (Glioma = 99, Normal = 10). (G) ARPC5 protein expression was detected in cerebral cortex (Patient id: 1582), low grade glioma (Patient id: 3365), and high-grade glioma (Patient id: 3251) tissues from HPA dataset. (H) Representative images of ARPC5 expression in glioma and its surrounding tissues. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Expressing, Quantitative Proteomics

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 2 Correlation between different status of ARPC5 expression and prognosis of glioma patients. (A) Survival analysis of ARPC5 in CGGA database. (B) ROC curve analysis of ARPC5 at 1, 3, and 5 years in CGGA database. (C) Survival analysis of ARPC5 in TCGA database. (D) ROC curve analysis of ARPC5 at 1, 3, and 5 years in TCGA database. Survival analysis of the signature in patients stratified by grade (E, F), gender (G, H), age (I, J), IDH mutation status (K, L), 1p/19q codeletion status (M, N), and MGMTp methylation status (O, P) in CGGA database

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Expressing, Mutagenesis, Methylation

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 3 Prognostic significance of ARPC5 and its association with drug sensitivity in glioma. (A) Univariate regression analysis of prognosis in CGGA data base. (B) Multivariate analysis of prognosis in CGGA database. (C) The nomogram to predict the association between ARPC5 expression and OS was de veloped using the CGGA dataset. (D) The calibration curve for the nomogram-predicted OS. ARPC5 affects the sensitivity to 5-fluorouracil (E), bleomycin (F), etoposide (G), crizotinib (H), sorafenib (I), and erlotinib (J)

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Expressing

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 4 Enrichment analysis identifies ARPC5-associated signaling pathways. (A) Heatmap for DEGs generated by compare the difference of ARPC5 ex pression in glioma from the CGGA dataset. (B) Volcano plots illustrated all DEGs. Barplot showing the GO (C) and KEGG (D) analysis for ARPC5 in glioma. (E) GO functional annotation of ARPC5 in glioma from GSEA. (F) KEGG pathway analysis of ARPC5 in glioma from GSEA.

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Protein-Protein interactions, Generated, Functional Assay

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 5 Investigations the expression of ARPC5 in glioma through single-cell analysis. (A) Through dimension reduction analysis of CGGA single-cell data, TSNE plot depicted that 6,148 cells were classified into 16 clusters. (B) The violin plot showed the expression of ARPC5 in each type of cluster. (C) Sixteen clusters were divided into 5 types of cells: astrocyte, macrophage, monocyte, epithelial and T cell. (D) The scatter plot shows the expression of ARPC5 in each cell

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Expressing, Single-cell Analysis

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 6 Correlations of ARPC5 expression with immunity-related indexes. (A) Violin plot showed the correlation between immune classification and tumor purity. The immune infiltration models of low- and high-ARPC5 were detected through ssGSEA methods in glioma from the TCGA database. (C) Violin plot showed the correlation between ARPC5 with stromal scores, immune scores, and ESTIMATE scores. (D) Lollipop showed the correlation between ARPC5 with infiltrating immune cells. Scatter plot showed the correlation between ARPC5 with TMB (E) and MSI (F). (G-J) Violin plot showed the correla tion between ARPC5 with immunotherapy

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Expressing

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 7 Effect of ARPC5 on the expression of CD3 and prognosis. (A) Representative images revealed the relationships between the ARPC5 expression and T cell marker CD3 in gliomas. (B) Survival analysis of ARPC5 in glioma chip ZL-BraG180sur01.

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Expressing, Marker

Journal: BMC cancer

Article Title: ARPC5 acts as a potential prognostic biomarker that is associated with cell proliferation, migration and immune infiltrate in gliomas.

doi: 10.1186/s12885-023-11433-w

Figure Lengend Snippet: Fig. 8 Effect of ARPC5 on the proliferation and migration of glioma cells. (A) The expression level of ARPC5 protein was detected in LN229 and U251 cells after shRNA interference, GAPDH was used to confirm equal protein loading. (B) RT-qPCR verified the expression efficiency of ARPC5 in LN229 and U251 cells after shRNA interference. Growth curve assessing the effect of ARPC5 on the proliferation of LN229 (C) and U251 (D) cells. (E) The representa tive image showed the clone formation ability of LN229 and U251 cells after transfection. (F) Transwell analysis was further used to examine the effect of ARPC5 on migration of LN229 and U251 cells, and the number of migrating cells was quantitatively analyzed. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: Glioma tissue samples and glioma chip ZLBraG180sur01 were incubated with ARPC5 antibody (16717-1-AP, proteintech, Wuhan, China) and CD3 antibody (17617-1-AP, proteintech, Wuhan, China).

Techniques: Migration, Expressing, shRNA, Quantitative RT-PCR, Transfection

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

doi: 10.1038/s41374-018-0178-5

Figure Lengend Snippet: [A] Cellular senescence was enhanced in BDL WT mice compared to WT mice but reduced in BDL α-CGRP −/− mouse liver compared to BDL WT mice, which was verified by SA-β-gal staining in liver sections (n=4, Orig., magnification, 40×; scale bar=100μm). [B-C] The mRNA expression of [B] p16, p21 and CCL2 and PAI-1 as well as [C] p16 protein levels were increased in total liver from BDL WT mice compared to WT mice but decreased in total liver from BDL α-CGRP −/− mice compared to BDL WT mice (n=3). * p <0.05 vs. WT mice; # p <0.05 BDL WT mice.

Article Snippet: The mouse antibody against p16 (sc-166760) was purchased from Santa Cruz Biotechnology.

Techniques: Staining, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

doi: 10.1038/s41374-018-0178-5

Figure Lengend Snippet: [A] The mRNA expression of α-SMA, Col1α1 and Fn1 was decreased in cholangiocytes from BDL α-CGRP −/− mice compared to BDL WT mice (n=4). [B] The mRNA expression of p16, p21 and PAI-1 was decreased in cholangiocytes from BDL α-CGRP −/− mice compared to BDL WT mice (n=4). [C] The mRNA expression of SASP markers such as IL-6, IL-8, IL1-β and MMP2 was decreased in cholangiocytes from BDL α-CGRP −/− mice compared to BDL WT mice (n=4). [D] Immunofluorescence demonstrated that p16 protein expression was decreased in cholangiocytes from BDL α-CGRP −/− mice compared to BDL WT mice (n=3, Orig. magnification, 20×, scale bar= 50 μm). * p 0.05 vs. WT mice; # p <0.05 vs. BDL WT mice.

Article Snippet: The mouse antibody against p16 (sc-166760) was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Immunofluorescence

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

doi: 10.1038/s41374-018-0178-5

Figure Lengend Snippet: [A] The mRNA expression of Col1α1 was decreased in HSCs isolated from BDL α-CGRP −/− mice compared to BDL WT mice (n=3). [B] The p16 and p21 mRNA expression was increased in BDL α-CGRP −/− mice compared to BDL WT mice (n=3). [C] Immunofluorescent staining showed that p16 protein expression was decreased in HSCs from BDL WT mice compared to BDL α-CGRP −/− mice (n=3, Orig. magnification, 40×, scale bar= 25 μm). # p <0.05 vs. BDL WT mice.

Article Snippet: The mouse antibody against p16 (sc-166760) was purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Isolation, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

doi: 10.1038/s41374-018-0178-5

Figure Lengend Snippet: [A] Representative immunofluorescence picture of receptor activity-modifying protein 1 (RAMP1) in HHSCs and IMCLs were shown (n=4, Orig., magnification. 40×; scale bar=50μm). [B-C] α-CGRP stimulated the expression of α-SMA and Col1 α 1 in both [B] IMCLs and [C] HHSCs, which was prevented by CGRP 8–37 (n=4). [D] The expression of p16 and p18 was decreased in HHSCs while increased in ICMLs simulated by α-CGRP; these effects were partly reversed by incubation with CGRP 8–37 (n=4). * p <0.05 vs. Basal; # p <0.05 vs. α-CGRP-treated group.

Article Snippet: The mouse antibody against p16 (sc-166760) was purchased from Santa Cruz Biotechnology.

Techniques: Immunofluorescence, Activity Assay, Expressing, Incubation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Knockout of α-calcitonin gene-related peptide attenuates cholestatic liver injury by differentially regulating cellular senescence of hepatic stellate cells and cholangiocytes

doi: 10.1038/s41374-018-0178-5

Figure Lengend Snippet: [A-C] The mRNA expression of [A] TGF-β1, [B] TIMP1 and [C ] Fn1 increased in HHSCs stimulated with cholangiocyte supernatant from BDL WT mice compared to HHSCs stimulated with cholangiocyte supernatant from WT mice but decreased in HHSCs treated with cholangiocyte from BDL α-CGRP −/− mice compared to HHSCs stimulated with cholangiocyte supernatant from BDL WT mice (n=4). [D-F] The mRNA expression of [D] p16, [E] p18 and [F] p21 was increased in HHSCs treated with cholangiocyte supernatant from BDL α-CGRP −/− mice compared to HHSCs stimulated with BDL cholangiocyte supernatant (n=4). * p <0.05 vs. HHSCs treated with cholangiocyte supernatant from WT mice; # p <0.05 vs. HHSCs treated with cholangiocyte supernatant from BDL WT mice.

Article Snippet: The mouse antibody against p16 (sc-166760) was purchased from Santa Cruz Biotechnology.

Techniques: Expressing

Journal: Oncotarget

Article Title: Establishment and characterization of a penile cancer cell line, penl1, with a deleterious TP53 mutation as a paradigm of HPV-negative penile carcinogenesis

doi: 10.18632/oncotarget.10098

Figure Lengend Snippet: The epithelial markers, pan-CK, CK5 and desmoplakin, were positive in LNM and xenografted tumors but negative in normal lymph nodes. Vimentin, a mesenchymal marker, was positive in a portion of LNM, normal lymph nodes and xenografted tumors. HPV 16/18 E6 was negative in all sections. Weak, diffuse and focally scattered positive staining for p16 was observed in LNM and xenografted tumors. Positive p53 nuclear expression was noted in LNM and xenografted tumors. Membrane expression of PDPN and EGFR was also positive in LNM and xenografted tumors.

Article Snippet: Primary antibodies against pan-CK, Vimentin, CK 5, desmoplakin, HPV16 E6/18 E6, p16 (Santa Cruz, USA), p53 rabbit mAb (Cell Signaling Technology, USA), and EGFR (Santa Cruz, USA) were used.

Techniques: Marker, Staining, Expressing, Membrane

Journal: Journal of Biological Chemistry

Article Title: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

doi: 10.1074/jbc.m115.657775

Figure Lengend Snippet: FIGURE 4. Nuclear CaMKII overexpression in CPCs promotes increases in cellular size and decreased proliferation. A, CPCeB has decreased proliferative capacity relative to CPCe based on a fluorescent nucleic acid stain measured at 2, 4, and 6 days after seeding equal cell numbers. ** and *** p 0.01 and p 0.0001 CPCeBrelativetoCPCe.B,CPCeBpopulationsshowsanincreaseddoublingtimerelativetoCPCe.C,cellcycleanalysisusingflowcytometryinCPCeandCPCeB.D, fluorescent images of CPCe and E, CPCeB populations in growth media. F, CPCeB shows an increase in relative cell surface area. ***, p 0.0001 CPCeB relative to CPC and CPCe. G, CPCeB shows an increase in length to width ratio relative to non-modified CPCs and CPCe. *, p 0.01 CPCeB relative to CPCe. H, expression of senescence markers p16 and p53 by immunoblot. -Actin was probed as a loading control. I, quantitation of p16 and J, p53 in CPCe and CPCeB populations.

Article Snippet: Next day blots were washed with TBST buffer and incubated in secondary antibodies in milk for 1.5 h. Primary antibodies for Western blot are as follows: rabbit anti-CaMKII (1:15000; UC Davis, Dr. Donald Bers), mouse anti-HDAC4 (1:1,000; Cell Signaling #5392) rabbit anti-phospho-HDAC4 (Ser-632)/HDAC5 (Ser-661)/ HDAC7 (Ser-486) (1:1000; Cell Signaling #3424) goat anti-GFP (1:1000; Rockland 600-101-215), mouse anti-HA tag (1:200; Santa Cruz Biotechnology, Inc. sc-7392), mouse anti-p16 (1:200, Santa Cruz Biotechnology, Inc. F-9 and F-12 clones sc-55600, sc-1661), mouse anti-p53 (1:500, Abcam ab26), rabbit anti- -sarcomeric actinin (1:500; Abcam ab72592), mouse anti- -smooth muscle actin (1:1000; Sigma Aldrich a2547), rabbit anti-Tie2 (1:100; Santa Cruz Biotechnology Inc. sc-9026), mouse anti-mouse -actin (1:500; Santa Cruz Biotechnology Inc sc-81178), rabbit anti-Mef2c (1:500; Cell Signaling #5030), rabbit anti-Cyclin B1 (1:500; Cell Signaling #12231), rabbit anti-Cyclin D1 (1:1000; Cell Signaling #2978), rabbit anti-phospho histone H3 (1:1000, Life Technologies 44 –1190G), mouse anti-GAPDH (1:3000; Millipore AB2302), and rabbit anti-Lamin A (C-terminal) (1:1000; Sigma Aldrich L1293).

Techniques: Over Expression, Staining, Modification, Expressing, Western Blot, Control, Quantitation Assay

Journal: Journal of Biological Chemistry

Article Title: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment

doi: 10.1074/jbc.m115.657775

Figure Lengend Snippet: FIGURE 9. Knockdown of CaMKIIB in CPCs prevents the up-regulation of pro-apoptotic molecules and promotes properties of cellular senescence. A, CaMKIIB; B, CaMKIIC; and C, Bcl-2 gene expression in CPC sh-Ctrl and CPC sh-B during growth conditions or with the addition of dex. Values are represented as n-fold mRNA relative to CPC sh-Ctrl in GM and after normalization to ribosomal 18S. **, p 0.01. D, proliferation measured using a direct nucleic acid fluorescent dye. Fluorescent values are represented as a fold change relative to day of plating (Day 0). E, cell doubling time in hours. F, cell cycle analysis using propidium iodide to determine percentage of cells in the different stages of the cell cycle analyzed by flow cytometry. G, cell cycle images of CPC sh-Ctrl and H. CPC sh-B. I, fluorescent images of CPC sh-Ctrl; and J, CPC sh-B. K, CPC sh-Ctrl and CPC sh-B cell surface area and L, length to width ratio represented as arbitrary units. M, immunoblot representation of pHDAC4 on the serine 632 and p16. N, quantitation of pHDAC4 and O. Senescence marker p16. Values are represented as a fold change relative to CPC sh-Ctrl and after normalizing to GAPDH.

Article Snippet: Next day blots were washed with TBST buffer and incubated in secondary antibodies in milk for 1.5 h. Primary antibodies for Western blot are as follows: rabbit anti-CaMKII (1:15000; UC Davis, Dr. Donald Bers), mouse anti-HDAC4 (1:1,000; Cell Signaling #5392) rabbit anti-phospho-HDAC4 (Ser-632)/HDAC5 (Ser-661)/ HDAC7 (Ser-486) (1:1000; Cell Signaling #3424) goat anti-GFP (1:1000; Rockland 600-101-215), mouse anti-HA tag (1:200; Santa Cruz Biotechnology, Inc. sc-7392), mouse anti-p16 (1:200, Santa Cruz Biotechnology, Inc. F-9 and F-12 clones sc-55600, sc-1661), mouse anti-p53 (1:500, Abcam ab26), rabbit anti- -sarcomeric actinin (1:500; Abcam ab72592), mouse anti- -smooth muscle actin (1:1000; Sigma Aldrich a2547), rabbit anti-Tie2 (1:100; Santa Cruz Biotechnology Inc. sc-9026), mouse anti-mouse -actin (1:500; Santa Cruz Biotechnology Inc sc-81178), rabbit anti-Mef2c (1:500; Cell Signaling #5030), rabbit anti-Cyclin B1 (1:500; Cell Signaling #12231), rabbit anti-Cyclin D1 (1:1000; Cell Signaling #2978), rabbit anti-phospho histone H3 (1:1000, Life Technologies 44 –1190G), mouse anti-GAPDH (1:3000; Millipore AB2302), and rabbit anti-Lamin A (C-terminal) (1:1000; Sigma Aldrich L1293).

Techniques: Knockdown, Gene Expression, Cell Cycle Assay, Flow Cytometry, Western Blot, Quantitation Assay, Marker