Fig. 6
From: Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane
Rif1 C466/C473 S-acylation mediates a peripheral DNA damage response. a Confocal microscopy of cells expressing Rif1NTD-GFP and Nup49-Ruby2, untreated or treated with Zeocin (100 μg/ml, 30 min; see Supplementary Fig. 6c, d for IR treatment). Z-projected images of cells in the indicated cell cycle phases, illustrating the observed classes of focus-negative and Rif1NTD focus-positive (1–4 foci) cells. For untreated cells, only the main class (focus-negative, representing ~75% of cells) is shown. Scale bar: 5 μm. b Quantification of focus-positive and focus-negative cells for the indicated strains expressing wild-type or mutant Rif1NTD-GFP, treated or not with Zeocin. Results of three independent experiments (n ≥ 100 cells per experiment) are presented as mean values ± s.e.m. For statistical analysis of GFP-positive cells treated or not with Zeocin, one-way Anova and a post-hoc Tukey–Kramer multiple comparison test was performed, comparing wild-type to mutants. c Quantitation of Rif1NTD-GFP foci per cell for the indicated strains with and without DNA-damage treatment (Zeocin, IR). Violin plots show data from three independent experiments (n ≥ 95 cells per strain and condition), binned for number of foci per cell. The average number of foci per cell (± s.e.m.) and the fraction of cells with ≥2 foci (± s.e.m.) are indicated. For statistical analysis, comparing wild-type to the indicated mutants with and without DNA-damage treatment, one-way Anova and a post-hoc Tukey–Kramer multiple comparison test was performed; unpaired t-test for cells treated or not with IR. d Zoning assay scoring the position of Rif1NTD-GFP foci in the indicated strains treated with Zeocin relative to the nuclear envelope (marked by Nup49-Ruby2). Foci were binned in three concentric zones of equal area; dashed line at 33% indicates random distribution. Results of three independent experiments (n = 50 Rif1 foci from 50 focus-positive cells per strain per experiment) are presented as mean values ± s.e.m. For statistical analysis, one-way Anova and a post-hoc Tukey–Kramer multiple comparison test was performed. Nuclear zones in wild-type were compared to the corresponding zones in the indicated mutants. See also Supplementary Fig. 6. Source data are provided as a Source Data file
