Distinct roles of VMP1 and TMEM41B in DMV biogenesis, related to Fig. 3. (A) TEM analysis shows that when nsp3/4 are ectopically expressed, large number of DMVs (white arrow) are induced in control COS7 cells. In VMP1 KO COS7 cells, the zippered ER forms concentric membrane structures (red arrow) and only a few DMVs are generated. N, nucleus. Bars: left panel, 500 nm; right panel, 200 nm. (B–D) In FPP assays, time-lapse images show that most of the nsp4-mCherry puncta persist upon pK addition in digitonin-permeabilized control COS7 cells coexpressing nsp3/4 (C). In VMP1 KO COS7 cells, nsp4–mCherry is quickly digested upon pK treatment (D). Quantitative data are shown as mean ± SEM (Ctrl, n = 11; VMP1 KO, n = 12; B). Bars: 5 μm. (E and F) In FPP assays, similar to control HeLa cells, mCherry-nsp3 is digested by pK, while nsp4-GFP is resistant to pK in TMEM41B KO cells expressing nsp3/4 (E). Quantitative data are shown as mean ± SEM (n = 18; F). Bars: 5 μm. (G) In FPP assays, nsp4-mCherry is sensitive to pK treatment in VMP1 KO HeLa cells expressing GFP-VMP1(G197R). Bar: 5 μm. (H) Immunostaining with LC3 antibody shows that GFP-nsp3/nsp4-mCherry double positive foci are separate from LC3 puncta. Bars: 5 μm; insert, 2 μm. (I) TEM analysis demonstrates that clusters of DMVs are formed in both control and FIP200 KO HeLa cells coexpressing nsp3/4. N, nucleus. Bars: top panel, 500 nm; bottom panel, 200 nm. (J) TEM analysis demonstrates that clusters of DMVs are formed in both control and TG-treated HeLa cells coexpressing nsp3/4. N, nucleus. Bars: 200 nm. (K) Immunostaining shows that Flag-nsp3/nsp4-mCherry double-positive foci are induced normally in FIP200 KO HeLa cells. Bars: 5 μm; insert, 2 μm. (L) Immunostaining shows that Flag-nsp3/nsp4-mCherry double-positive foci are induced normally in TG-treated HeLa cells. TG, thapsigargin. Bars: 5 μm; insert, 2 μm.