Supplementary Figure 2: Galleries of postmitotic NPC assembly intermediates and quantification of membrane distance and curvature.

(a) Other images of pre-pores at different times after AO (4.8, 6.1, 7.7, 10, and 15 min). ONM, outer nuclear membrane; INM, inner nuclear membrane. Scale bar, 100 nm. (b, c) Quantification of the distance between ONM and INM (b) and the radius of membrane tip curvature (c) as indicated by a blue bidirectional arrow and a red arrow in the left panels. The plots of the ONM/INM distance and the tip curvature are from 71, 75, 88, 64, 58, and 52 pores at 4.8, 6.1, 7.7, 10, 15 min, and in interphase, respectively. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers. A blue line shows a fitted sigmoid curve. For comparison, a sigmoid curve of pre-pore diameter in Fig. 2c is shown by a pink dashed line. Interestingly, the distance between ONM and INM was very highly variable during early stages of NPC assembly, and became constrained from 36 to 25 nm from 10 min onwards only after pore dilation was completed (b), suggesting that the linker of nucleoskeleton and cytoskeleton (LINC) complex, which connects ONM and INM (Shimi, T., Butin-Israeli, V. & Goldman, R. D. Curr. Opin. Cell Biol. 24, 71–78, 2012), is established after NPC assembly is completed. On the other hand, the tip curvature of the pre-pore membrane was constant at all time points (c), implying the continuous association of membrane-shaping protein(s) such as reticulons followed by Nups155 and 53 during NPC assembly³.