FXRs are essential for forming and maintaining gathered DMVs. (A) Western blotting shows the KD efficiency of siFXRs. (B and C) Fluorescence images reveal numerous small Nsp3/4⁺ puncta in cells after KD of all FXR genes, while a few big Nsp3/4⁺ puncta are formed in control cells (B). Numbers of large and all Nsp3/4⁺ puncta are shown as mean ± SD (NC, n = 30; siFXRs, n = 30) in C. ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (D–F) TEM images show that dispersed DMVs are observed in siFXRs cells, while clustered DMVs are present in NC-treated cells (D). Percentages of cells with clustered DMVs (at least five DMVs gathering together) are quantified in E, and numbers and sizes of DMVs are shown as mean ± SD in F (NC, n = 85; siFXRs, n = 99). ****, P < 0.0001; N.S., not significant. Bars: 500 nm. (G and H) Re-expressing RNAi-resistant GFP-FXR1, but not GFP, induces big Nsp3/4⁺ puncta in siFXRs cells (G). Numbers of large and all Nsp3/4⁺ puncta are shown as mean ± SD (NC, n = 30; GFP, n = 25; GFP-FXR1, n = 27) (H). ****, P < 0.0001; N.S., not significant. Bars: 5 μm; insets, 2 μm. (I–K) Big Nsp3/4⁺ puncta are formed in cells coexpresssing mCherry-Nsp3 and Nsp4-GFP for 2 days (I). When the cells are treated with NC or siFXRs, big Nsp3/4⁺ puncta remain present in NC cells while a large number of Nsp3/4⁺ dots are induced after KD of FXRs (J). Numbers of large and all Nsp3/4⁺ puncta are shown as mean ± SD (NC, n = 34; siFXRs, n = 41) (K). ****, P < 0.0001. Bar: 5 μm; insets, 2 μm. Source data are available for this figure: SourceData F2.