Sir3 overexpression specifically leads to Rap1-GFP foci clustering. (A) Growth assay and telomeric silencing assay at telVIIL::URA3. To assess the toxicity of SIR overexpression, wild-type (WT; yAT232), GAL1p-SIR2 (yAT200), GAL1p-SIR3 (yAT208), and GAL1p-SIR4 (yAT202) cells were grown in glucose, and fivefold serial dilutions were plated either onto YPD or YPGal plates. To monitor telomeric silencing at telVIIL, strains were grown in galactose synthetic medium for 48 h and then plated onto 5-FOA galactose plates. Decreased growth on YPGal plates indicates SIR overexpression toxicity, and decreased growth on 5-FOA plates reflects a disruption of telomeric silencing. (B) Rap1 foci grouping upon Sir3 overexpression. Representative fluorescent images of the telomere-associated protein Rap1 tagged with GFP of the strains used in A. Cells were grown in galactose overnight, diluted to OD_600nm = 0.2, and imaged at OD_600nm = 1. Numbers represent the mean intensity of foci. Bar, 2 µm. (C) Quantification of images from B using an application developed in house (Q-Foci; see Materials and methods). Gray levels are set to represent the distribution of foci intensity in wild-type cells. (D) Levels of Rap1 are stable upon Sir3 overexpression. Immunoblots with anti-GFP and anti-Sir3 on crude extracts from wild type (yAT7; first lane), GAL1p-SIR3 (yAT370) in repressive conditions (second lane), and after 3 h of Sir3 induction (third lane). (E) Graphical representation of the foci intensity as a function of the number of foci per cells (data from C) in a wild type and in a strain overexpressing Sir3. Horizontal bars represent the median for each category of cells.