Lipins and nuclear LDs. (A) qRT-PCR. The lipin mRNA levels are expressed relative to β-actin mRNA. A representative result of two independent experiments. (B) GFP-lipin distribution in U2OS cultured in normal medium, with OA for 1 d or with OA for 1 d and OA and 0.25 µM Torin1 (OA/Torin1) for another 8 h. The pie charts show the relative distribution of GFP–lipin-1α and GFP–lipin-1β in cells treated with OA and Torin1. (C) The effect of Torin1. U2OS cultured with OA for 2 d or treated with OA for 1 d and OA/Torin1 for another day. The number of nuclei counted: 30. Pooled data from three independent experiments. Mann–Whitney test; ***, P < 0.001. (D) Distribution of GFP–lipin-1α and -1β (green; arrowheads) in cells cultured with OA for 1 d and with OA/Torin1 for another 8 h. LDs, magenta. (E) The effect of wild-type and mutant lipin-1β expression. U2OS depleted of lipin-1 was transfected with siRNA-resistant GFP–lipin-1 cDNAs and cultured with OA for 1 d and with OA/Torin1 for another 8 h. The number of nuclei counted: 49 (GFP), 58 (WT), 56 (D714E), or 44 (LxxFF). Pooled data from three independent experiments. Box boundaries indicate the 25th and 75th percentiles, and whiskers indicate Tukey type. Kruskal–Wallis ANOVA test followed by Dunn’s test; *, P < 0.05. Scale bars, 10 µm.