Extended Data Fig. 9: Spartin-DN impairs LD turnover in SUM159 cells.
From: The Troyer syndrome protein spartin mediates selective autophagy of lipid droplets
a, Confocal imaging of live SUM159 wild-type (top panel) and spartin KO (bottom panel) cells transiently expressing mScarlet-I-tagged spartin-DN or spartin full-length (Spartin-FL) proteins. LDs were stained with BODIPY493/503. Transfected cells were marked with yellow outline. Scale bars: 20 μm. b, Quantification of LD numbers per cell shown in (a). Mean ± SD, n = 58 cells (WT+mSc-I), 39 cells (WT+Spartin-DN), 37 cells (WT+Spartin-FL), 39 cells (Spartin KO+Spartin-DN), 33 cells (Spartin KO+Spartin-DN), and 30 cells (Spartin KO+Spartin-FL) from three independent experiments, n.s., not significant, ****p < 0.0001, one-way ANOVA, Dunnett’s multiple comparisons test. c, SUM159 wild-type cells transiently expressing mScarlet-I or mScarlet-I-spartin DN were pulse-labeled with [14C]-OA, and incorporation into TG was measured after treatment with 0.5 mM OA for 30 and 60 min. Values were calculated relative to cells expressing mScarlet-I highest value at 60 min. Mean ± SD, n = 6 independent experiments, n.s. not significant, two-way ANOVA, multiple comparisons test. d, Overlay live-cell confocal images of Keima–LiveDrop acquired by 488 nm (green) and 561 nm (magenta) excitations collected with a 607/36 nm emission filter, expressed in SUM159 wild-type, transiently transfected with Halo–spartin WT and DN constructs as described. Scale bars: 20 μm. e, Ratiometric fluorescence measurement of Keima–LiveDrop per cell shown in (d). Mean ± SD, n = 12 cells from three independent experiments, ****p < 0.0001, two-tailed unpaired t-test. f, Overlay live-cell confocal images of Keima–spartin-WT and Keima–spartin-DN acquired by 488 nm (green) and 561 nm (magenta) excitations collected with a 607/36 nm emission filter, expressed in SUM159 wild-type cells. g, Ratiometric fluorescence measurement of Keima signal per cell shown in (f). Mean ± SD, n = 14 cells from three independent experiments, ****p < 0.0001, two-tailed unpaired t-test. h, Confocal imaging of live SUM159 cells transiently expressing LAMP1-mNG and mScarlet-I–spartin-WT (top) or mScarlet-I–spartin-DN (bottom). Cells were treated with 0.5 mM OA for 24 h and chased for 3 h after OA withdrawal. Scale bars: full-size, 20 μm; insets, 2 μm. Source numerical data are available in source data.
