Extended Data Fig. 7: ERES increase in number upon LD induction and are required for late ER-to-LD protein targeting.
From: Identification of two pathways mediating protein targeting from ER to lipid droplets
a, Sec16 and Tango1 are required for LD targeting of Ldsdh1. Confocal imaging of wildtype cells upon RNAi of ERES components, followed by transient transfection with Ldsdh1-EGFP and a 20-hr incubation in oleate-containing medium. Scale bar, 5 and 1 μm (inlay). Mean ± SD, n = (left to right) 79, 41, 52 cells examined over 3 independent experiments. One-way ANOVA with Bonferroni correction, #p < 0.0001, compared to LacZ. b, Sec16 and Tango1 are required for LD targeting of HSD17B11. Scale bar, 5 and 1 μm (inlay). Bar graph shows percentage of cells with LD targeting (defined as those with >2 LDs with protein targeting). Mean ± SD, n = 3 experiments except 6 for LacZ RNAi (14–17 cells each). One-way ANOVA with Bonferroni correction, #p < 0.0001, compared to LacZ. c, d, Bar graph showing percentage of cells for a given number of LDs with HSD17B11-EGFP targeting upon RNAi of ERES components. Representative images in b. e, f, Sec16 and Tango1 are dispensable for LD targeting of (e) LDAH and Ubxd8 and (f) Lsd1, CGI-58, and CCT1. Scale bar, 5 and 1 μm (inlay). For mCherry-CCT1, nuclear signal was excluded from the calculation. Mean ± SD, n = (left to right) LDAH 87, 36, 36; Ubxd8 85, 44, 43; Lsd1 79, 39, 41; CGI-58 74, 36, 38; CCT1 54, 26, 31 cells examined over 3 independent experiments. One-way ANOVA with Bonferroni correction, *p < 0.05 (from top to bottom: 0.0159, 0.0193), compared to LacZ. g, LD induction transiently increases the number of Sec16 and Tango1 puncta. Representative images in Fig. 4e. Mean ± SD, n = (left to right) 38, 39, 33, 37; 38, 39, 35, 37 cells examined over 2 independent experiments. One-way ANOVA with Bonferroni correction, ***p < 0.001 (from left to right: 0.0001, 0.0008), #p < 0.0001. h, Localization of Sec16 around LDs and its recruitment of endogenous Tango1 require Sec12 and Sar1 but not Sec23. Representative images from 3 independent experiments are shown. Scale bar, 5 and 1 μm (inlay). i, Overexpressed Sar1-H74G and Sec23 accumulate around LDs upon Tango1 depletion. Representative images from 5 independent experiments are shown. Scale bar, 5 and 1 μm (inlay). Source numerical data are available in source data.
