Extended Data Fig. 6: The co-localization of shieldin with RIF1 on chromatin.
From: The shieldin complex mediates 53BP1-dependent DNA repair
a, Representation of the deletion mutants of SHLD2-N used in c, d. The orange shading indicates blocks of homology. b, Schematic of the LacR–RIF1 chromatin recruitment assay. c, Quantification of the experiment shown in d. Colocalization was considered positive when the average GFP intensity at the mCherry focus was threefold over background nuclear intensity. A minimum of 20 cells were imaged per biological replicate (circles); the bar represents the mean ± s.d. (n = 3). d, Representative images of the data quantified in c. The main focus is shown in inset ; scale bar, 10 μm. e–h, Quantification (e, g) and representative micrographs (f, h) of overexpressed GFP–SHLD2-N and mCherry–LacR–RIF1(1–967) co-transfected into uninduced U2OS–FokI cells along with siRNA against shieldin complex subunits after processing for mCherry and GFP (e, f) or mCherry and REV7 (g, h) immunofluorescence. Colocalization was considered positive when the average GFP or REV7 intensity at the mCherry focus was threefold over background nuclear intensity. A minimum of 20 cells were imaged per condition (circles); the bar represents the mean ± s.d. (n = 3 biologically independent experiments). i, Representative images of the data quantified in j. The main focus is shown in inset; scale bar, 10 μm. j, Quantification of GFP intensity at the mCherry–LacR–RIF1(1–967) focus, normalized to nuclear background. Each data point represents a cell transfected with the vector coding for the indicated GFP fusion. The line is at the median. The data are an aggregate of three independent experiments with a minimum of 20 cells counted (total cells counted: 62, 60 and 61 for GFP, GFP–SHLD2-C and GFP–SHLD3, respectively). k, mCherry–LacR–FokI colocalization with full-length or N-terminally truncated (Δ1–50) GFP–SHLD2. Mean normalized focus intensity is shown from a total of 59 (full-length SHLD2) or 56 (SHLD2 Δ1–50) cells counted (n = 2 biologically independent experiments).
