Characterization of nuclear LDs in U2OS. (A) Electron micrograph of U2OS cultured with OA for 1 d. Arrow indicates an LD in the nucleoplasm. The nuclear envelope and the ER lumen are delineated by DAB precipitated by HRP-KDEL. Scale bar, 0.2 µm. (B) Colocalization of PML (red) and nuclear LDs (green). (Upper panel) Endogenous PML was immunolabeled. (Lower panel) mCherry PML-II was expressed. Nucleus, blue. Scale bars, 10 µm; 2 µm (magnified photos). (C) Western blotting of MTP and actin. Total lysates of Huh7, A549 (human alveolar adenocarcinoma), and U2OS were cultured with or without OA supplementation. (D) BiFC. Green fluorescence due to complementation between GFP_1–10 and RFP-GFP₁₁ is present in both the nucleus and the cytoplasm, whereas that of NLSx3-GFP_1–10 and RFP-GFP₁₁ is confined to the nucleus. Scale bar, 10 µm. (E) BiFC with GFP_1-10. GPAT3, GPAT4, LPCAT1, and DGAT1 tagged with GFP₁₁ produce fluorescence signal in the cytoplasmic network. Nucleus, blue. Scale bar,10 µm. (F) Immuno-EM of GFP-ACSL3 using ultrathin cryosections. Immunogold labels in the INM (arrowheads). Scale bar, 0.2 µm. (G) Immunolabeling of endogenous ACSL3 in nuclear LDs (arrowhead). LD, green; nucleus, blue. Scale bar, 10 µm. (H) Comparison of GFP-DGAT2 and GFP-PK-DGAT2. (i) Distribution of GFP-PK-DGAT2 (green). LDs, magenta. Arrowheads indicate nuclear LDs. Scale bar, 10 µm. See Fig. 2 B for distribution of GFP-DGAT2. (ii) The expression level of GFP-DGAT2 and GFP-PK-DGAT2 (Western blotting).