Extended Data Fig. 5: Synchronization of ER-to-LD protein transport using the RUSH system.
From: Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets
a. Quantification of SBP-LiveDrop targeting kinetics after biotin-induced release. Targeting ratio was calculated as the intensity of SBP-LiveDrop within the BODIPY-stained LD mask normalized to total cellular SBP-LiveDrop intensity and baseline (pre-release) levels. Ncells = 35. Shaded area represents standard deviation. b. SBP-LiveDrop targeting ratio to LDs measured at multiple time points post-biotin release and displayed in a boxplot. The box represents the 25th–75th percentiles (IQR), the center line indicates the median. Statistical significance was assessed by a two-sided unpaired t-tests: P = 0.004 (0 vs 0.5 h), 0.009 (0 vs 3 h), 0.0001 (0 vs 24 h), 0.86 (0.5 vs 3 h), 0.04 (0.5 vs 24 h), 0.08 (3 vs 24 h). c. Representative confocal images showing SBP-LiveDrop localization to nascent and mature LDs after biotin release. Cells were pretreated with 250 µM oleic acid for varying durations to induce LD formation, followed by a 30-minute biotin release prior to imaging. LDs were stained with 1 µM BODIPY 493/503. Scale bar: 10 µm (top), 2 µm (bottom). d. Quantification of SBP-LiveDrop intensity normalized to LD area, calculated by dividing the targeting ratio by the total BODIPY mask area per image, reflecting SBP-LiveDrop density on LD surfaces after 30 minutes of biotin release. Time 0 represents no OA addition. Error bars show standard deviation. Values for each field of view (N = 7) are overlaid on graphs. Statistical significance was assessed by unpaired t-tests with p-values indicated for comparisons between time points in OA. P = 0.33, Ncells = 23 (0 vs 1 h); P = 0.11, Ncells = 29 (0 vs 2 h); P = 0.73, Ncells = 26 (0 vs 5 h); P = 0.21, Ncells = 32 (0 vs 24 h). e. Total LD area (left) and SBP-LiveDrop targeting ratio (right) quantified after 30 minutes of biotin release in cells incubated with 250 µM oleic acid for increasing durations. Time 0 represents no OA addition. Error bars show standard deviation. Values for each field of view (N = 7) are overlaid on graphs. Statistical significance was assessed by unpaired t-tests with p-values indicated for comparisons between time points in OA. P = 0.000052, Ncells = 23 (0 vs 1 h); P = 0.000048, Ncells = 29 (0 vs 2 h); P = 1.3 × 10−6, Ncells = 26 (0 vs 5 h); P = 5.9 × 10−9, Ncells = 32 (0 vs 24 h). Source numerical data are available in source data.
