Extended Data Fig. 10: LPCAT3 activity does not affect gross membrane lipid-packing indices.

(a) Shotgun lipidomic analysis of PC composition in ER or LD–ER-enriched fractions from Lpcat3^KO adipocytes expressing Flag-LPCAT3^WT or Flag-LPCAT3^H374A and differentiated for 6 days. Data are presented as the distribution of PC species (% of total PC) within each sample. (b) Schematic depiction of the trilayer model system containing a bulk oil phase flanked by phospholipid monolayers, solvated with water (left), and top/side views of trilayers exhibiting LPDs that expose the oil core to the solvent (right). (c) Deep/shallow LPDs in model bilayers and LD-like trilayer systems (± 3–5% surface tension, ST). Each replicate corresponds to a block-averaged LPD value derived from individual MD simulation trajectories (n = 3/group). WT and Lpcat3^KO compositions reflect complex (left) and simplified (right) lipidomic approximations (Supplementary Table 6). (d,e) Immunoblot analysis of Lpcat3^KO adipocytes expressing Flag-LPCAT3^WT or Flag-LPCAT3^H374A and treated with (d) ± isoproterenol (ISO, 10 µM), or (e) ± insulin (100 nM) for 15 min. Actin and Calnexin served as sample processing controls. (f) Tandem mass tag (TMT) labelling on 8,000g LDs isolated from Lpcat3^KO adipocytes expressing Flag-LPCAT3^WT or Flag-LPCAT3^H374A (day 6 of differentiation). TMT data were overlaid with Uniprot mouse proteome for annotated LD localization. (g) Top/side snapshots from CG–MD simulations of an ER–LD neck system. Bilayer/monolayers consist of 50% PUFA (cyan) and 50% MUFA (white) phospholipids, with a triolein (TO; orange) core. (h) Electron microscopy analysis of Lpcat3^KO adipocytes expressing Flag-LPCAT3^WT or Flag-LPCAT3^H374A (day 3 of differentiation). (i) Lipolysis in Lpcat3^KO and Lpcat3/Atgl^DKO adipocytes expressing Flag-LPCAT3^WT or Flag-LPCAT3^H374A. Secreted glycerol was measured after ISO (10 µM) treatment for 2 h, normalized to intracellular TG stores (n = 3 wells/group). Where indicated, cells were differentiated in the presence of 0.1% DMSO (veh ctrl) or Atglistatin (ATGLi; 40 µM) for 6 days. Data are presented as mean ± SEM. Protein abundance ratios were compared across groups using Student’s t-test (P < 0.01) while controlling for false-discovery rate with Benjamini-Hochberg procedure (f); or ***P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test (i).

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