Fig. 1. Principles of DBSCAN and hierarchical clustering. (a) DBSCAN; (b) hierarchical clustering

Fig. 2. Flowchart of NPC SMLM data processing

Fig. 3. Screening of NPC. (a) Simulated distribution map of NPCs; (b) segmentation of NPCs and fine division of Nups; (c) keeping NPCs with 4 to 8 Nups; (d) filtering NPCs by shape factor of outline polygons; (e) finally selected NPCs that meet the screening criteria

Fig. 4. Reconstruction of NPC. (a)‒(c) A schematic diagram depicting a standard orientation rotation of an NPC composed of 7 Nups: (a) initial state; (b) clockwise rotation δ1 around the center of the NPC from initial state; (c) clockwise rotation δiaround the center of the NPC from initial stage. (d) Alignment of all the screened NPC centers. (e) Eightfold symmetric structure after rotational alignment

Fig. 5. Reconstruction and analysis of Nup133 SMLM images. (a)(b) Typical SMLM image of Nup133 in U2OS cells and its zoomed view; (c) remained NPCs with 4‒8 Nup133s after preliminary screening; (d) finally selected NPCs in accordance with the screening criteria; (e) histogram of Nup133 number distribution; (f) spatial distribution of Nup133 positions aligned to NPC centers; (g) distribution of Nup133 radius, in which the red line represents the Gaussian-fitted distribution curve and its horizontal ordinate of the peak is the fitted Nup133 radius; (h) spatial distribution of Nup133 after rotational alignment

Fig. 6. Reconstruction and analysis of Nup98 SMLM images. (a)(b) Typical SMLM image of Nup98 in U2OS cells and its zoomed view; (c) remained NPCs with 4‒8 Nup98s after preliminary screening; (d) finally selected NPCs in accordance with the screening criteria; (e) histogram of Nup98 number distribution; (f) spatial distribution of Nup98 positions aligned to NPC centers; (g) distribution of Nup98 radius, in which the red line represents the Gaussian-fitted distribution curve and its horizontal ordinate of the peak is the fitted Nup98 radius; (h) the distribution of Nup98 after rotational alignment; (i) model diagram of NPC; (j) overlay of Nup133 and Nup98 distributions