Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: Cav-1 levels increase as cells reach senescence. ( A ) Characteristic Immunoblot showing caveolin-1 (Cav-1) levels increasing significantly as WJ-MSCs reach senescence. β-Actin was used as control for equal protein loading. ( B ) Representative photos of early-, middle- and late-passage cells stained with SA-β-gal staining solution. ( C ) Graph showing the percentage of SA-β-gal positive cells in early-, middle- and late-passage cells as mean from three donors. * p < 0.05 vs. NT or otherwise indicated. p values calculated using the Student t -test. ( D ) Representative images of early- and late-passage WJ-MSCs where total ROS and superoxide levels were analyzed under normal conditions (early–late). For a positive control pyocyanine was added to the early passage cells (early + PYO). Images were captured with the 40X objective lens of the fluorescent microscope used.

Article Snippet: Oxidative stress was detected by staining with two fluorescent dyes from the ROS-ID ® Total ROS/Superoxide detection kit (ENZ-51010, Enzo, Farmingdale, NY, USA).

Techniques: Western Blot, Control, Staining, Positive Control, Microscopy

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: Late-passage cells demonstrated an inability to repair DNA damage. ( A ) Characteristic images of early- and late-passage cells where DNA damage was assessed by staining with the 53BP1 antibody and the appropriate secondary antibody (green). (blue). Cav-1 localization was also evaluated by staining with anti-cav-1 antibody and the appropriate secondary antibody (red) under normal conditions (No Treatment—NT) and at 1 h and 24 h after being treated with H 2 O 2 . (300 μΜ, 30 min). Nuclei were stained with DAPI. Images were captured with the 40× objective lens of the fluorescent microscope used. ( B ) Percentages of WJ-MSCs with 53BP1 foci from early- and late-passage cells from three donors evaluated by staining with the 53BP1 antibody under normal conditions (No Treatment—NT) and at different time points of the recovery period from the oxidative treatment (1 h and 24 h). Values shown are the means ± S.E. # p < 0.05 vs. early passage cells at the same time point and * p < 0.05 vs. NT or otherwise indicated. p values calculated using the Student t -test.

Article Snippet: Oxidative stress was detected by staining with two fluorescent dyes from the ROS-ID ® Total ROS/Superoxide detection kit (ENZ-51010, Enzo, Farmingdale, NY, USA).

Techniques: Staining, Microscopy

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: No alterations in Cav-1/pCav-1 levels in late-passage cells after exogenous oxidative insult. ( A ) Representative immunoblot of cell lysates of early- and late-passage WJ-MSCS (from three donors) that have been exposed to H 2 O 2 (300 μΜ, 30 min) and left to recover for 24 h, analyzed at four different time points (NT(=No Treatment), 1 h, 3 h, and 24 h of recovery time post-treatment) by western blotting using the anti-caveolin-1 (Cav-1) antibody. ( B ) Quantification of relative protein expression levels of Cav-1 in cell lysates from early and late WJ-MSCs (from 3 donors) after being exposed to H 2 O 2 was performed based on band density using ImageJ. NT values from both early- and late-passage cells were arbitrarily set to 1. Values shown are the means ± S.E. * p < 0.05 vs. the NT condition. ( C ) Characteristic images of early- and late-passage cells where Cav-1 localization was evaluated by staining with anti-cav-1 antibody and the appropriate secondary antibody (red) under normal conditions (No Treatment—NT) and 24 h after being treated with Doxorubicin. DNA damage was also assessed by staining with the 53BP1 antibody and the appropriate secondary antibody (green). Nuclei were stained with DAPI (blue). Images were captured with the 40× objective lens of the fluorescent microscope used. ( D ) Percentages of WJ-MSCs with 53BP1 foci from early- and late-passage cells from three donors evaluated by staining with the 53BP1 antibody under normal conditions (No Treatment—NT) and 24 h after Doxorubicin treatment. Values shown are the means ± S.E. # p < 0.05 vs. early passage cells at NT and * p < 0.05 vs. NT or otherwise indicated. p values calculated using the Student t -test. ( E ) Representative immunoblot of cell lysates of early- and late-passage WJ-MSCS (from 3 donors) that have been exposed for 24 h to Doxorubicin using anti-caveolin-1 (Cav-1) antibody. ( F ) Quantification of relative protein expression levels of Cav-1 in cell lysates from early and late WJ-MSCs (from 3 donors) after being exposed to 24 h Doxorubicin was performed based on band density using ImageJ. NT values from both early- and late-passage cells were arbitrarily set to 1. Values shown are the means ± S.E. * p < 0.05 vs. the NT condition. ( G ) Representative immunoblot showing p-Cav-1 levels from cell lysates of early- and late-passage WJ-MSCS (from three donors) that have been exposed to H 2 O 2 (300 μΜ, 30 min) and left to recover for 24 h, analyzed at three different time points ((NT(=No Treatment), 1 h and 24 h of recovery time post-treatment). Antibody against β-Actin was used as loading control for all western blots (A, B, D). ( H ) Quantification of relative protein expression levels of p-Cav-1 in cell lysates from early and late WJ-MSCs (from three donors) after being exposed to H 2 O 2 was performed based on band density using ImageJ. Values shown are the means ± S.E. * p < 0.05 vs. the NT condition and # p < 0.05 vs. early passage cells. p values were calculated using Student t -test.

Article Snippet: Oxidative stress was detected by staining with two fluorescent dyes from the ROS-ID ® Total ROS/Superoxide detection kit (ENZ-51010, Enzo, Farmingdale, NY, USA).

Techniques: Western Blot, Expressing, Staining, Microscopy, Control

Journal: Cells

Article Title: Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset

doi: 10.3390/cells10112939

Figure Lengend Snippet: Cav-1 downregulation results in failure of oxidative DNA damage repair. ( A ) Characteristic immunoblot showing caveolin-1 (Cav-1) levels decreasing significantly after siRNA against Cav-1 treatment in early passage cells. Antibody against β-Actin was used as loading control. ( B ) Graph demonstrating Cav-1 levels based on band density calculated using Image J. Values shown are the means ± S.E. * p < 0.05 vs. the early passage WJ-MSCs treated with control siRNA (siCTRL). ( C ) Characteristic images of early passage cells treated with control siRNA and siRNA against Cav-1, under normal conditions (No Treatment—NT) and at 1 and 24 h after being treated with H 2 O 2 (300 μΜ, 30 min) stained with anti-cav-1 antibody and the appropriate secondary antibody (red). DNA damage was also assessed by staining with the 53BP1 antibody and the appropriate secondary antibody (green). Nuclei were stained with DAPI (blue). Images were captured with the 40× objective lens of the fluorescent microscope used. ( D ) Percentages of cells with 53BP1 foci from early passage WJ-MSCs treated with siCTRL and siCav-1 under normal conditions (No Treatment—NT) and at 1 h and 24 h after being treated with H 2 O 2 (300 μΜ, 30 min). Values shown are the means ± S.E. # p < 0.05 vs. siCTRL treated early passage cells and * p < 0.05 vs. NT or otherwise indicated. p values calculated using the Student t -test.

Article Snippet: Oxidative stress was detected by staining with two fluorescent dyes from the ROS-ID ® Total ROS/Superoxide detection kit (ENZ-51010, Enzo, Farmingdale, NY, USA).

Techniques: Western Blot, Control, Staining, Microscopy

Journal:

Article Title: Immunoassays and Biosensors for Monitoring Environmental and Human Exposure to Pyrethroid Insecticides

doi: 10.1021/jf1033569

Figure Lengend Snippet: Fluorescent immunoassay for 3-PBA using iron-cored luminescent Tb shell nanoparticles. (a) The competitive hapten-linked protein conjugate was immobilized to the luminescent particle, and competed with 3-PBA to bind to specific antibody. After removing unbound immunocomplex by magnetic separation, the reporter FITC-labeled secondary antibody was added for quantification; (b) fluorescent signals of organic dyes (FITC or TRITC) as reporters and luminescent nanoparticles as a reference; (c) scanning of fluorescent signals of the luminescent nanoparticles complexed with the FITC-labeled antibody against various concentrations of 3-PBA in the range of 450 nm-550 nm, excited at 270 nm; (d) the standard curve represents the 3-PBA concentration dependence of the ratio between the fluorescence intensity of the FITC dye reporter (IFITC) and the intensity of the magnetic luminescent Tb particles (ITb).

Article Snippet: We are working on various immunoassay technologies such as fluorescent resonance energy transfer (FRET) and fluorescent polarization using immunoreagents such as antibodies, phage displayed peptides, competitive hapten mimics, magnetic luminescent nanoparticles, and its paired fluorescent dyes.

Techniques: Labeling, Concentration Assay, Fluorescence