Figure 4.
Seipin and nuclear LDs.(A) The effect of seipin RNAi. U2OS were transfected with control siRNA, seipin siRNA, or seipin siRNA and ACSL3 siRNA and cultured with OA for 1 d. The number of nuclei counted: 48 (control), 48 (seipin), 43 (seipin+ACSL3). Pooled data from three independent experiments. Kruskal–Wallis ANOVA test followed by Dunn’s test; ***, P < 0.001. (B) Re-expression of seipin in seipin-deficient cells. U2OS depleted of seipin was transfected with nontagged seipin cDNA and GFP cDNA (10:1 ratio) and cultured with OA for 1 d. GFP, magenta; LD, green; nucleus, blue. The number of nuclei counted: 134 (GFP), 110 (GFP+seipin). Pooled data from three independent experiments. Box boundaries indicate the 25th and 75th percentiles, and whiskers indicate Tukey type. Mann–Whitney test; **, P < 0.01. (C) EM of U2OS transfected with seipin siRNA and cultured with OA for 1 d. The nuclear envelope and the ER lumen are delineated by DAB precipitated by HRP-KDEL. Nuclear LDs, arrowheads. (D) BiFC. Both NLSx3-RFP-GFP1-10 and RFP-GFP1-10 are used for GFP11-seipin. (E) Immuno-EM of GFP-seipin in ultrathin cryosections. Immunogold labels in the outer nuclear membrane (ONM; arrowheads) and the ER (arrows). (F) FRAP of GFP-ACSL3 in nuclear and cytoplasmic LDs. U2OS transfected with either control or seipin siRNA cultured with OA for 1 d. The number of analyzed LDs: 14 (control) or 15 (seipin) for nuclear LDs; 39 (control) or 29 (seipin) for cytoplasmic LDs. Recovery plots show the mean ± SEM and the curve fitting with nonlinear regression and the exponential one-phase decay model. The pie charts show the proportion of LDs showing >40% (purple) or <40% (gray) fluorescence recovery 15 min after photobleaching. See Videos 7 and 8 for representative results. (G) Super-resolution microscopy of nuclear LDs (mRuby3-NLSx3-HPos) and the INM (GFP-Lap2β) in U2OS transfected with either control or seipin siRNA and cultured with OA for 1 d. The distance between nuclear LDs and the INM was measured; n = 70 (control), 138 (seipin). Pooled data from three independent experiments. Mann–Whitney test; **, P < 0.01. The proportion of nuclear LDs within 0.1 µm of the INM: 2.9% for control, 15.2% seipin. Scale bars, 10 µm (fluorescence micrographs); 0.2 µm (EM).

Seipin and nuclear LDs. (A) The effect of seipin RNAi. U2OS were transfected with control siRNA, seipin siRNA, or seipin siRNA and ACSL3 siRNA and cultured with OA for 1 d. The number of nuclei counted: 48 (control), 48 (seipin), 43 (seipin+ACSL3). Pooled data from three independent experiments. Kruskal–Wallis ANOVA test followed by Dunn’s test; ***, P < 0.001. (B) Re-expression of seipin in seipin-deficient cells. U2OS depleted of seipin was transfected with nontagged seipin cDNA and GFP cDNA (10:1 ratio) and cultured with OA for 1 d. GFP, magenta; LD, green; nucleus, blue. The number of nuclei counted: 134 (GFP), 110 (GFP+seipin). Pooled data from three independent experiments. Box boundaries indicate the 25th and 75th percentiles, and whiskers indicate Tukey type. Mann–Whitney test; **, P < 0.01. (C) EM of U2OS transfected with seipin siRNA and cultured with OA for 1 d. The nuclear envelope and the ER lumen are delineated by DAB precipitated by HRP-KDEL. Nuclear LDs, arrowheads. (D) BiFC. Both NLSx3-RFP-GFP1-10 and RFP-GFP1-10 are used for GFP11-seipin. (E) Immuno-EM of GFP-seipin in ultrathin cryosections. Immunogold labels in the outer nuclear membrane (ONM; arrowheads) and the ER (arrows). (F) FRAP of GFP-ACSL3 in nuclear and cytoplasmic LDs. U2OS transfected with either control or seipin siRNA cultured with OA for 1 d. The number of analyzed LDs: 14 (control) or 15 (seipin) for nuclear LDs; 39 (control) or 29 (seipin) for cytoplasmic LDs. Recovery plots show the mean ± SEM and the curve fitting with nonlinear regression and the exponential one-phase decay model. The pie charts show the proportion of LDs showing >40% (purple) or <40% (gray) fluorescence recovery 15 min after photobleaching. See Videos 7 and 8 for representative results. (G) Super-resolution microscopy of nuclear LDs (mRuby3-NLSx3-HPos) and the INM (GFP-Lap2β) in U2OS transfected with either control or seipin siRNA and cultured with OA for 1 d. The distance between nuclear LDs and the INM was measured; n = 70 (control), 138 (seipin). Pooled data from three independent experiments. Mann–Whitney test; **, P < 0.01. The proportion of nuclear LDs within 0.1 µm of the INM: 2.9% for control, 15.2% seipin. Scale bars, 10 µm (fluorescence micrographs); 0.2 µm (EM).

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