Htl1 depletion causes abnormal NPC morphology and nuclear envelope stress. (a) HTL1 shut-off strains expressing the indicated GFP-tagged NPC proteins were grown in the presence of glucose and imaged at the indicated times. Maximum-intensity projections of representative large-budded cells are shown. Bar, 3 μm. Corresponding flow cytometry histograms can be found in Fig. S5 a. (b) Nic96- and Nup170-GFP intensity per pixel was measured as a proxy for NPC morphology. At least 100 HTL1 shut-off cells were measured for each strain. Glucose shut-offs were performed in the absence (vector) and presence of NBP1 overexpression. The distribution of GFP intensity per pixel is displayed, as is the percentage of pixels above and below 100,000 arbitrary units. The P values from a one-tailed Mann-Whitney test are shown. Corresponding flow cytometry histograms can be found in Fig. S5 b. (c) NPC structure (red triangles) was examined using TEM on an HTL1 shut-off strain after 12 h of logarithmic growth in media containing glucose. A representative section showing eight NPCs on a nuclear tubule is shown. (d) TEM was performed on an HTL1 shut-off strain after 12 h of logarithmic growth in media containing glucose. Both images represent sections from the same large-budded cell. An abnormal nuclear flare (red arrow; left) and a monopolar SPB (red arrow; right) can be seen. Bar, 0.5 μm.