Extended Data Figure 7. Analysis of GFP-TONSL localization.

a, Co-localization analysis of chromatin-bound GFP-TONSL with MCM2 analysed by deconvolution microscopy and measurement of Pearson coefficient in single cells. Error bars, SD, n=13 from two independent experiments. Representative image, Fig. 3c. b, c, Representative images for the analysis shown in Fig. 3d. Cells were either pulsed with EdU (40 μM) for 15 minutes (b) or synchronized in G1/S and released into S phase in continuous presence of EdU (5 μM) (c). Images are representative of b: n=9 (very early), 16 (early/mid), 10 (mid/late); c: 9 (very early), 27 (early/mid), 36 (mid/late). Scale bar, 5 μm. (b) EdU and MCM2 staining was used to determine the cell cycle state of asynchronous cells. (c) Progression through S phase was followed by FACS analysis of DNA content. d, Chromatin-binding of GFP-TONSL analysed by cellular fractionation in inducible U-2-OS cells as quantified in Fig. 3e. S, soluble. C, chromatin. e, f, Chromatin-binding analysis as in Fig. 3f. U-2-OS cells conditional for GFP-TONSL ARD WT and mutant were directly fixed or pre-extracted to remove soluble proteins. Data are representative of 3 (e) and 2 (f) experiments, fields of cells in (e) are representative of (from left) n=16, 18, 17 and 17 images. Scale bar, 20 μm. g, Asynchronous U-2-OS cells conditional for GFP-TONSL were pulsed with 40 μM EdU for 15 minutes and soluble proteins were extracted. Representative images of EdU positive cells are shown (n=30 for WT and N571A), for the specific patterns of TONSL WT see Fig. 3d. Scale bar, 5 μm.