Journal: Biogerontology

Article Title: Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae

doi: 10.1007/s10522-014-9499-y

Figure Lengend Snippet: Relocation of Rap1p ( a ) and Sir3p ( b ) from telomeres to the nucleolus and changes in expression patterns of Rap1p, Sir3p and Nop2p ( c ) during chronological aging. a, b After 2, 7 and 14 days, Rap1p ( a ) and Sir3p ( b ) immunostaining was performed ( green ). DNA was visualised using DAPI staining ( blue ). Immunostained cells were captured with an Olympus BX61 light microscope equipped with a DP72 CCD camera and Olympus CellF software. Nop2p was used as a nucleolar marker ( red ). Typical micrographs of haploid wild type BY4741 are shown. c After 2 and 7 days, changes in the expression of Rap1p, Sir3p and Nop2p were revealed with western blotting. For the loading control, an antibody against actin was used. The chemiluminescence signal was detected using an ECL Plus Western Blotting Detection System (GE Healthcare) and G:BOX imaging system (Syngene). Top haploid strains, bottom diploid strains. (Color figure online)

Article Snippet: Spheroplast-coated slides were blocked with 1 % BSA in TBST (20 mM Tris–HCl, pH 7.5, 137 mM NaCl containing 0.1 % Tween 20) at room temperature for 30 min and: Nop2p was detected using a mouse monoclonal antibody against Nop2p (diluted 1:1,000 in 1 % BSA in TBST, overnight incubation at 4 °C) (Santa Cruz Biotechnology, Germany) and anti-mouse Texas Red-conjugated antibody (diluted 1:500 in 1 % BSA in TBST, 1 h incubation at room temperature) (Santa Cruz Biotechnology, Germany); Rap1p was detected using a goat polyclonal antibody against Rap1p (diluted 1:200 in 1 % BSA in TBST, overnight incubation at 4 °C) (Santa Cruz Biotechnology, Germany) and anti-goat FITC-conjugated antibody (diluted 1:500 in 1 % BSA in TBST, 1 h incubation at room temperature) (Santa Cruz Biotechnology, Germany); Sir3p was detected using a rabbit polyclonal antibody against Sir3p (diluted 1:200 in 1 % BSA in TBST, overnight incubation at 4 °C) (Santa Cruz Biotechnology, Germany) and anti-rabbit FITC-conjugated antibody (diluted 1:500 in 1 % BSA in TBST, 1 h incubation at room temperature) (Santa Cruz Biotechnology, Germany).

Techniques: Expressing, Immunostaining, Staining, Light Microscopy, Software, Marker, Western Blot, Control, Imaging

Journal: Biogerontology

Article Title: Links between nucleolar activity, rDNA stability, aneuploidy and chronological aging in the yeast Saccharomyces cerevisiae

doi: 10.1007/s10522-014-9499-y

Figure Lengend Snippet: rDNA instability-based chronological aging in yeast. During CA, oxidative stress-induced chromosome XII instability may contribute to both rDNA instability and whole chromosome aneuploidy. Moreover, altered redox equilibrium may promote oxidative protein modifications leading to changes in the protein expression patterns, turnover and functions. Breaks within rDNA are subjected to DNA repair processes, e.g., by homologous recombination (HR) and/or non-homologous end joining (NHEJ), which may result in altered nucleolar architecture and activity (nucleolus enlargement, rDNA accumulation). Finally, CA-mediated nucleolus fragmentation may be a consequence of nucleolus enlargement and/or Nop2p upregulation

Article Snippet: Spheroplast-coated slides were blocked with 1 % BSA in TBST (20 mM Tris–HCl, pH 7.5, 137 mM NaCl containing 0.1 % Tween 20) at room temperature for 30 min and: Nop2p was detected using a mouse monoclonal antibody against Nop2p (diluted 1:1,000 in 1 % BSA in TBST, overnight incubation at 4 °C) (Santa Cruz Biotechnology, Germany) and anti-mouse Texas Red-conjugated antibody (diluted 1:500 in 1 % BSA in TBST, 1 h incubation at room temperature) (Santa Cruz Biotechnology, Germany); Rap1p was detected using a goat polyclonal antibody against Rap1p (diluted 1:200 in 1 % BSA in TBST, overnight incubation at 4 °C) (Santa Cruz Biotechnology, Germany) and anti-goat FITC-conjugated antibody (diluted 1:500 in 1 % BSA in TBST, 1 h incubation at room temperature) (Santa Cruz Biotechnology, Germany); Sir3p was detected using a rabbit polyclonal antibody against Sir3p (diluted 1:200 in 1 % BSA in TBST, overnight incubation at 4 °C) (Santa Cruz Biotechnology, Germany) and anti-rabbit FITC-conjugated antibody (diluted 1:500 in 1 % BSA in TBST, 1 h incubation at room temperature) (Santa Cruz Biotechnology, Germany).

Techniques: Expressing, Homologous Recombination, Non-Homologous End Joining, Activity Assay