Figure S3.
Effects of seipin knockdown on PA in the nucleus.(A) Western blotting showing the efficacy of seipin/lipin-1 double knockdown. (B) Colocalization of mCherry-GPAT4 (magenta) and NLS-Opi1 Q2-GFP (green). Scale bar, 10 µm. (C) The effect of OA/Torin1 on NLS-Opi1 Q2-GFP (green). U2OS expressing NLS-Opi1 Q2-GFP or NLS-Opi1 Q2mut-GFP were cultured with OA for 1 d and OA/Torin1 for another 8 h. The long-exposure image shows the invariable association of NLS-Opi1 Q2-GFP signals (arrowheads) with nuclear LDs (magenta). Scale bars, 10 µm. (D) The number of PA puncta in the nucleus. U2OS expressing NLS-Opi1 Q2-GFP (Opi1 Q2) was cultured either in normal medium, in medium with OA for 1 d, or in medium with OA for 1 d and with OA/Torin1 for another 8 h. Cells expressing NLS-Opi1 Q2mut-GFP (Opi1 Q2mut) were treated with OA and OA/Torin1. The number of nuclei counted: 30 (Opi1 Q2), 15 (Opi1 Q2mut). Pooled data from three (Opi1 Q2) or two (Opi1 Q2mut) independent experiments. Kruskal–Wallis ANOVA test followed by Dunn’s test; **, P < 0.01; ***, P < 0.001; n.s., not significant. (E) Live imaging of NLS-Opi1 Q2-GFP (green) in U2OS treated with OA for 1 d. LDs, red. Selected frames from Video 9 are shown. Arrows indicate a nuclear LD with fluctuating PA signals. Scale bar, 2 µm. (F) The effect of seipin overexpression on the number of PA puncta in the nucleus. The number of nuclei counted: 18. A representative result of two independent experiments. Welch’s unpaired t test; **, P < 0.01. (G) Western blotting for verification of nuclear isolation. Equal amounts of proteins were loaded for the total cell and isolated nucleus samples. (H) The effect of GPAT3/4 overexpression. (Hi) Large cytoplasmic LDs surrounded with mCherry-tagged GPAT3 and GPAT4 (arrowheads). Scale bar,10 µm. (Hii) Nuclear LDs. The number of nuclei counted: 18 (mCherry), 23 (mCherry-GPAT3), 20 (mCherry-GPAT4). A representative result of two independent experiments. Mann–Whitney test. (Hiii) Nuclear PA puncta. The number of nuclei counted: 20. A representative result of two independent experiments. Mann–Whitney test. (I) The effect of perilipin-3 knockdown on PC synthesis in U2OS. Incorporation of 3H-choline to PC was measured 3 d after perilipin-3 siRNA transfection. A representative result of two independent experiments.

Effects of seipin knockdown on PA in the nucleus. (A) Western blotting showing the efficacy of seipin/lipin-1 double knockdown. (B) Colocalization of mCherry-GPAT4 (magenta) and NLS-Opi1 Q2-GFP (green). Scale bar, 10 µm. (C) The effect of OA/Torin1 on NLS-Opi1 Q2-GFP (green). U2OS expressing NLS-Opi1 Q2-GFP or NLS-Opi1 Q2mut-GFP were cultured with OA for 1 d and OA/Torin1 for another 8 h. The long-exposure image shows the invariable association of NLS-Opi1 Q2-GFP signals (arrowheads) with nuclear LDs (magenta). Scale bars, 10 µm. (D) The number of PA puncta in the nucleus. U2OS expressing NLS-Opi1 Q2-GFP (Opi1 Q2) was cultured either in normal medium, in medium with OA for 1 d, or in medium with OA for 1 d and with OA/Torin1 for another 8 h. Cells expressing NLS-Opi1 Q2mut-GFP (Opi1 Q2mut) were treated with OA and OA/Torin1. The number of nuclei counted: 30 (Opi1 Q2), 15 (Opi1 Q2mut). Pooled data from three (Opi1 Q2) or two (Opi1 Q2mut) independent experiments. Kruskal–Wallis ANOVA test followed by Dunn’s test; **, P < 0.01; ***, P < 0.001; n.s., not significant. (E) Live imaging of NLS-Opi1 Q2-GFP (green) in U2OS treated with OA for 1 d. LDs, red. Selected frames from Video 9 are shown. Arrows indicate a nuclear LD with fluctuating PA signals. Scale bar, 2 µm. (F) The effect of seipin overexpression on the number of PA puncta in the nucleus. The number of nuclei counted: 18. A representative result of two independent experiments. Welch’s unpaired t test; **, P < 0.01. (G) Western blotting for verification of nuclear isolation. Equal amounts of proteins were loaded for the total cell and isolated nucleus samples. (H) The effect of GPAT3/4 overexpression. (Hi) Large cytoplasmic LDs surrounded with mCherry-tagged GPAT3 and GPAT4 (arrowheads). Scale bar,10 µm. (Hii) Nuclear LDs. The number of nuclei counted: 18 (mCherry), 23 (mCherry-GPAT3), 20 (mCherry-GPAT4). A representative result of two independent experiments. Mann–Whitney test. (Hiii) Nuclear PA puncta. The number of nuclei counted: 20. A representative result of two independent experiments. Mann–Whitney test. (I) The effect of perilipin-3 knockdown on PC synthesis in U2OS. Incorporation of 3H-choline to PC was measured 3 d after perilipin-3 siRNA transfection. A representative result of two independent experiments.

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