NOMO depletion disrupts ER architecture and dynamics. (A) Spinning disc confocal microscopy images of U2OS cells under NOMO knockdown (siNOMO), showing z-stack projections at +3 µm from the basal plane shown at the left. Scale bars: 10 µm. (B) Transmission electron microscopy (TEM) images of control (siNT) and NOMO knockdown (siNOMO) U2OS cells. Arrows (black) denote ER; white arrowheads denote membrane delineated structures “voids” arising upon NOMO depletion. Scale bars: 1 µm. (C and D) Immunofluorescence images of HeLa cells under either non-targeting (siNT) or NOMO knockdown (siNOMO) conditions with calreticulin and the indicated cytoskeletal stain. (E) Quantification of void area in WT U2OS cells across conditions (Ctrl, WT NOMO, and 4-Mut NOMO). Data represent mean ± SD. Statistical significance was determined by an ordinary one-way ANOVA test; ns, not significant. (F–H) Superplots of FRAP data for WT NOMO, 4-Mut NOMO, and Δ10–11 NOMO constructs, with multiple biological replicates (Runs) displayed with a minimum of 10 cells per run. Each plot represents normalized fluorescence intensity over time after bleaching. (I) Table of FRAP recovery tau values (τ, in seconds) for indicated NOMO constructs across multiple experimental runs from E. (J) FRAP analysis of ER-localized Sec61β -GFP in control (Ctrl) and NOMO knockdown (siNOMO) cells. Source numerical data are available in source data.