Loss of Brr6 and Brl1 cause NPC assembly defects. (A) Serial-dilution growth assay of td-brr6 td-brl1 cells. (B) Images of living cells incubated at 37°C for 3 h. The yeGFP signal along the NE was scanned (enlargement right, yellow circle) for the distribution of the NUPs (graph, bottom). Arrowheads indicate GFP-dots in the cytoplasm. A cartoon of NUPs with GFP-fusions used in B and in Fig. S1 D is shown. Bars: (overviews) 5 µm; (enlargements) 1 µm. (C) Percentage of cells with mislocalization of GFP-fusion proteins. Error bars: SD (n > 180); three independent experiments. (D) TEM micrographs of BRR6 BRL1 WT and td-brr6 td-brl1 cells incubated for 3 h at 37°C. Anti-Nsp1 staining at normal NPCs (asterisk), herniations (arrowheads), and sheets. N, nucleus; C, cytoplasm. Bars, 100 nm. (E) RITE assay. Estradiol induces a genetic switch of NUP188-mCherry (old) to NUP188-yeGFP (new). td-brr6 td-brl1 cells were imaged at 10-min intervals after addition of estradiol at 37°C. Fig. S1 I shows an additional td-brr6 td-brl1 cell. Bar, 1 µm.