Extended Data Fig. 3: Validation of 2D analysis of 3D ER structure.

(a) 3D projection view of a U2OS cell expressing sec61-mCherry. The peripherical ER is mainly distributed as a flat, single layer while the perinuclear ER is more 3 dimensional in character (see Supplementary Video 12). (b) 3D projection view of a COS-7 cell expressing mEmerald-Sec61b-C1. i: (left) 3D reconstruction of image sections. ii: projection of the volumetric view of corresponding the sections shown in (i). Peripherical ER features are flat and singly layered (Supplementary Video 13). In the perinuclear region, multiple layers of ER are visible. iii: each section from the 3D image stack shown in (i) is segmented individually by ERnet. iv: The resulting segmented frames are then combined into a 3D volume to show the network topology (Supplementary Video 14). Cyan regions (tubules) show flat, 2D regions with little 3D character. Perinuclear regions appear in yellow (sheets), where the topology is 3 dimensional in character.