The effect of seipin knockdown. (A) Western blotting. U2OS transfected with control or seipin siRNA. (B) qRT-PCR. U2OS transfected with control or seipin siRNA. The lipin-1/β-actin mRNA ratio in the control sample is set to one. A representative result of three independent experiments. (C) The effect of seipin/lipin-1 double knockdown. U2OS transfected with seipin siRNA alone or with seipin siRNA and lipin-1 siRNA was cultured with OA for 1 d. The number of nuclei counted: 39 (seipin) or 40 (seipin+lipin-1). Pooled data from three independent experiments. Mann–Whitney test; ***, P < 0.001. (D) The effect of seipin knockdown on NLS-Opi1 Q2-GFP (green). U2OS were cultured with OA for 1 d. LD, magenta. The number of PA puncta/rings per nucleus was counted. The number of nuclei counted: 81 (control) or 57 (seipin). Pooled data from three independent experiments. Box boundaries indicate the 25th and 75th percentiles, and whiskers indicate Tukey type. Mann–Whitney test; ***, P < 0.001. (E) The effect of seipin knockdown on nuclear PA. PA and PC were quantified in nuclei isolated from U2OS cultured with OA for 2 d. A representative result of two independent experiments. (F) The effect of seipin knockdown on GFP–lipin-1β concentration in nuclear LDs. U2OS were cultured with OA for 1 d. Wild-type and PA phosphatase-deficient mutant (D714E) were compared. The number of nuclei counted: 60 (control siRNA+WT cDNA), 63 (seipin siRNA+WT cDNA), 41 (seipin siRNA+D714E cDNA). A summary of three independent experiments. (G) The effect of seipin/lipin-1 double knockdown on NLS-Opi1 Q2-GFP. The number of nuclei counted: 32 (seipin) or 34 (seipin+lipin-1). Pooled data from three independent experiments. Mann–Whitney test. (H) Diagram of nuclear LD formation. Nuclear LDs in normal cells will also form using nuclear PA and lipin-1. Scale bars, 10 µm. n.s., not significant.