Figure 1. Nup88, Nup98 and Sec13 are required for relocalization of heterochromatic DSBs.

(A) Schematic representation of nuclear pore proteins including chromatin-associated nucleoporins. Red indicates nucleoporins whose depletion affects relocalization/anchoring of heterochromatic DSBs from this study and12. (B) Immunofluorescence (IF) analysis and quantification of Kc cells fixed 60 min after IR show the number of γH2Av foci in DAPI-bright and total foci following indicated RNAi depletions. *P<0.05, **P<0.01, ****P<0.0001, n>195 cells for each RNAi condition. (C) As described in b, except cells expressing Nup96-MycFLAG were used. **P<0.01, ****P<0.0001, n>328 cells for each RNAi condition. (D) Chromatin fractionation and Western Blot (Wb) analysis of Kc cells shows the chromatin-bound fractions of Nup88, Nup98, and Sec13 at indicated time points after IR. Histone H4 and Ponceau are loading controls. (E) IF analysis shows γH2Av foci colocalizing with the indicated nucleoporins in DAPI-bright, at different timepoints after IR. Dashed boxes and zoomed details highlight colocalizations 10 min after IR. Kc cells were used for Nup88 and Sec13 staining. GFP-Nup98FG-expressing cells were used in place of Nup98 WT due to a stronger nucleoplasmic signal associated with this mutant. n>118 foci for each time point. Error bars: SEM in E, and SD in B,C from three or more independent experiments. P values calculated with two-tailed Mann–Whitney test. Images are max intensity projections of Z-stacks spanning the DAPI-bright region. Ctrl = Control. Scale bar = 1μm.