Workflow of cryo-CLIEM of NSP3/NSP4-induced DMVs, confocal analysis of the co-localization of different truncations of NSP3/NSP4 in HeLa cells, and TEM images of DMV in HEK-293T cells, related to Fig. 1 . (A) Co-localization signals of fluorescent GFP-nsp3 and nsp4-mCherry were observed in the vitrified HeLa cells, serving as a marker for the identification of regions containing DMVs. Scale bar, 20 μm. (A1) FIB image of the corresponding area in panel A. The blue arrow indicates the target cell, corresponding to the white box in A. (B) Cryo-FM image of the pre-lamella (∼400 nm) via FIB milling direction. Scale bar, 10 μm. (C) Cryo-FM image of the final lamella (∼150 nm). Scale bars, 10 μm. (D) The final lamella viewed from FIB milling direction. (E) The SEM image of the final lamella. (F and G) The cryo-EM image of the lamella, containing the DMVs of interest (white-dotted frame). LD, lipid droplet; ER, endoplasmic reticulum; NPC, nuclear pore complex; Mito, mitochondrion. Scale bar, 2 μm (F), 1 μm (G). (H) Confocal microscopy analysis of the co-localization of different truncations of NSP3/NSP4 in HeLa cells. Scale bar, 10 μm. (I) Conventional TEM images of DMV in HEK-293T cells induced by GFP-NSP3-T2A-NSP4(WT)-mCherry and GFP-NSP3-T2A-NSP4(ΔCTD)-mCherry. Scale bar, 500 nm. (J) Statistical analysis of DMV numbers per cell. N = 3, n = 16, 17 (N indicate the number of independent experiments and n indicate the number of total measurements or observations). All data are shown as mean ± SEM, Student’s two-tailed t test, *: P < 0.05, significant.