Journal: Antioxidants

Article Title: The microRNA Let-7f Induces Senescence and Exacerbates Oxidative Stress in Retinal Pigment Epithelial Cells

doi: 10.3390/antiox13060646

Figure Lengend Snippet: Let-7f induces a senescence phenotype in human retinal pigment epithelial cells. ARPE-19 cells were transfected with 50 nM let-7f mimic for 48 h and senescence was examined through various assays. ( A ) Proliferating cells were identified via immunofluorescent staining of Ki67 (green) positive cells and nuclei (DAPI, blue) (10×). ( B ) The percent of Ki67 positive cells was calculated based on the number of Ki67 positive cells divided by the total number of cells (DAPI positive) and expressed versus scramble control (Sc). ( C ) Representative images of senescence-associated β-galactosidase (SA-β-gal) staining used to detect senescent ARPE-19 cells. Senescent cells were identified by the development of blue coloration and an enlarged, flattened morphology as indicated by the red arrows (10×). ( D ) Percentage of senescent ARPE-19 cells was compared to scramble control (Sc). ( E ) The protein expression levels of cyclin-dependent kinase inhibitors p16 INK4a and p21 Waf/Cip1 were detected via western blotting. Band densitometry was calculated using ImageLab Software and normalized to β-actin. Results were presented as a fold change compared to scramble control cells (Sc). * p < 0.05, *** p < 0.001 vs. Sc. ns: not significant. All data are expressed as the mean ± SD ( n = 3).

Article Snippet: Membranes were blocked in 5% BSA for 2 h and incubated with mouse anti-human p16 INK4a (1:500, Lifespan Biosciences, Shirley, MA, USA), mouse anti-human p21 Waf/Cip1 (1:500, Lifespan Biosciences) or rabbit anti-human β-actin (1:5000, Novus Biologicals, Centennial, CO, USA) at 4 °C overnight.

Techniques: Transfection, Staining, Control, Expressing, Western Blot, Software

Journal: PLoS ONE

Article Title: Histone Demethylase Jumonji D3 ( JMJD3 ) as a Tumor Suppressor by Regulating p53 Protein Nuclear Stabilization

doi: 10.1371/journal.pone.0051407

Figure Lengend Snippet: Effect of JMJD3 overexpression on p16/p14, p53 and p21 mRNA expression in GSC 923 (wild-type INK4A/ARF and TP53 ) ( A ) and GSC 1228 ( ∧∧ INK4A/ARF null, wild-type TP53 ) ( B ) and HEK 293 cells (wild-type INK4A/ARF and TP53 ) ( C ). D , Western blot analysis showing the effect of JMJD3 on p21 and GFAP protein expression in primary GSCs and HEK 293 cells. All experiments were done in triplicates. Error bars represent means ±SD. p = two-tailed Student’s t test comparing indicated samples to empty vector control, *p<0.1, **p<0.05, ***p<0.01, N.S.-not significant.

Article Snippet: Primary anti-bodies used were as follows: mouse anti-human p53 IC12 (Cell Signaling 2524) 1∶1000, mouse anti-Flag (Sigma F3165) 10 μg/ml, mouse anti-human p21 WAF/CIP1 12D1 (Cell Signaling 2947S) 1∶1000, rabbit anti-human GFAP (Dako M0761) 1∶4000, rabbit anti-human histone 3 (Millipore 04–928) 1∶250, mouse anti-human tubulin (Sigma T3526) 1∶5000, For immunocytochemistry, mouse anti-Flag-FITC Conjugated (Sigma F4049) 10 μg/ml, rabbit anti-human H3K27me3 (Millipore 07–449) 1∶500, monoclonal mouse anti-β-tubulin III 1∶1000 (Covance MMS-435P), polyclonal rabbit anti-β-tubulin III 1∶1000 (Convance PRB-435P) and rabbit anti-human GFAP (Dako M0761) 1∶4000.

Techniques: Over Expression, Expressing, Western Blot, Two Tailed Test, Plasmid Preparation, Control

Journal: PLoS ONE

Article Title: Histone Demethylase Jumonji D3 ( JMJD3 ) as a Tumor Suppressor by Regulating p53 Protein Nuclear Stabilization

doi: 10.1371/journal.pone.0051407

Figure Lengend Snippet: A, Western blot analysis showing the effect of JMJD3 overexpression on total p53 levels in HEK 293 cells. B, Nuclear fractionation following JMJD3 overexpression in HEK 293 cells (N-Nuclear fraction, C-Cytoplasmic fraction). Total Histone 3 (Total H3) was used as nuclear loading control and Tubulin as cytoplasm loading control. Densitometry values of p53 expression (black) are relative to respective nuclear or cytoplasmic control. C, Schema of human p21 promoter showing p53 consensus binding sites and primers used for Chromatin immunoprecipitation with PCR (ChIP-PCR). TSS is transcription start site, 3′ and 5′ sites represent consensus p53 binding sites within the human p21 promoter. D–F, ChIP-PCR results showing localization of RNA polymerase II ( D ), p53 ( E ) and JMJD3 ( F ) at the p21 promoter in HEK 293 cells following JMJD3 overexpression. All experiments were done in triplicates. Error bars represent means ±SD. p = two-tailed Student’s t test comparing indicated samples to empty vector controls, *p<0.1, **p<0.05, ***p<0.01, N.S.- not significant.

Article Snippet: Primary anti-bodies used were as follows: mouse anti-human p53 IC12 (Cell Signaling 2524) 1∶1000, mouse anti-Flag (Sigma F3165) 10 μg/ml, mouse anti-human p21 WAF/CIP1 12D1 (Cell Signaling 2947S) 1∶1000, rabbit anti-human GFAP (Dako M0761) 1∶4000, rabbit anti-human histone 3 (Millipore 04–928) 1∶250, mouse anti-human tubulin (Sigma T3526) 1∶5000, For immunocytochemistry, mouse anti-Flag-FITC Conjugated (Sigma F4049) 10 μg/ml, rabbit anti-human H3K27me3 (Millipore 07–449) 1∶500, monoclonal mouse anti-β-tubulin III 1∶1000 (Covance MMS-435P), polyclonal rabbit anti-β-tubulin III 1∶1000 (Convance PRB-435P) and rabbit anti-human GFAP (Dako M0761) 1∶4000.

Techniques: Western Blot, Over Expression, Fractionation, Control, Expressing, Binding Assay, Chromatin Immunoprecipitation, Two Tailed Test, Plasmid Preparation

Journal: PLoS ONE

Article Title: Histone Demethylase Jumonji D3 ( JMJD3 ) as a Tumor Suppressor by Regulating p53 Protein Nuclear Stabilization

doi: 10.1371/journal.pone.0051407

Figure Lengend Snippet: Effect of JMJD3 overexpression on human p21 promoter luciferase reporter ( A ) in HEK 293 ( B ), mouse neural stem cells E14 (MsNSCs) ( C ) and GSC 923 ( D ) with a wild-type p53 5′ binding site (wild-type 5′ site) and mutant p53 5′ binding site (mutant 5′ site; Position 2279 C>A in red ( A )). TSS is transcription start site, 3′ and 5′ sites represent consensus p53 binding sites within the human p21 promoter. All experiments were done in triplicates. Error bars represent means ±SD. p = two-tailed Student’s t test comparing indicated samples, *p<0.1, **p<0.05, ***p<0.01, N.S.-not significant.

Article Snippet: Primary anti-bodies used were as follows: mouse anti-human p53 IC12 (Cell Signaling 2524) 1∶1000, mouse anti-Flag (Sigma F3165) 10 μg/ml, mouse anti-human p21 WAF/CIP1 12D1 (Cell Signaling 2947S) 1∶1000, rabbit anti-human GFAP (Dako M0761) 1∶4000, rabbit anti-human histone 3 (Millipore 04–928) 1∶250, mouse anti-human tubulin (Sigma T3526) 1∶5000, For immunocytochemistry, mouse anti-Flag-FITC Conjugated (Sigma F4049) 10 μg/ml, rabbit anti-human H3K27me3 (Millipore 07–449) 1∶500, monoclonal mouse anti-β-tubulin III 1∶1000 (Covance MMS-435P), polyclonal rabbit anti-β-tubulin III 1∶1000 (Convance PRB-435P) and rabbit anti-human GFAP (Dako M0761) 1∶4000.

Techniques: Over Expression, Luciferase, Binding Assay, Mutagenesis, Two Tailed Test