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. 2024 Jul 1;223(9):e202311126. doi: 10.1083/jcb.202311126

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© 2024 Li et al.

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Figure 4. — Low affinity splitFAST is suitable for implementing FABCON. (A) Distribution of mApple-6xHp and Halo-ER (top), NFAST_high-mApple-6xHp and CFAST-Halo-ER (middle), and NFAST_low-mApple-6xHp and CFAST-Halo-ER (bottom) in an oleic acid (OA)-treated U2OS cell. Representative images from a single axial plane are shown. (B) Quantification of fluorescence recovery after photobleaching of Halo-ER in the endoplasmic reticulum (ER) and near lipid droplets (LDs); or CFAST-Halo-ER in the ER and near NFAST_high- or NFAST_low-decorated LDs. Mean ± SD is shown (20–25 regions from three independent experiments). n.s. = not significant, ***P ≤ 0.001, unpaired t test, two-tailed. (C) Quantification of the Pearson’s colocalization coefficient of LDs and ER described in A. Raw data and mean ± SD are shown (11–16 cells from three independent experiments). **P ≤ 0.01, assessed by one-way ANOVA. (D) Quantification of the Pearson’s colocalization coefficient of LDs and mitochondria (mito) in cells expressing Halo-6xHp and NFAST_high-mApple-mito, CFAST-Halo-6xHp and NFAST_high-mApple-mito, or CFAST-Halo-6xHp and NFAST_low-mApple-mito (see Fig. S1 A). Raw data and mean ± SD are shown (29–32 cells from three independent experiments). ***P ≤ 0.001, assessed by one-way ANOVA. (E) Quantification of the Pearson’s colocalization coefficient of LDs and peroxisomes described in cells producing Halo-6xHp and PMP34-mApple, CFAST-Halo-6xHp and PMP34-mApple-NFAST_high, or CFAST-Halo-6xHp and PMP34-mApple-NFAST_low (see Fig. S1 B). Raw data and mean ± SD are shown (44–51 cells from three independent experiments). ***P ≤ 0.001, assessed by one-way ANOVA. (F) Quantification of the length of mito-LD contact sites detected by scanning transmission electron microscopy (STEM) in control HeLa cells or cells expressing FAB^mito-LD in the absence, 3 min after the addition, and 5 min after the washout of 3 µM HBR-2,5DOM. Raw data and mean ± SD are shown (n = 23 in control; n = 50 in N_low+CFAST without dye; n = 43 in N_low+CFAST with dye; n = 23 in N_low+CFAST after washout). n.s. = not significant, assessed by one-way ANOVA. (G) Representative electron micrographs of mito-LD contact sites detected by STEM in HeLa cells expressing FAB^mito-LD in the absence and presence of 3 µM HBR-2,5DOM for 3 min. (H) AlphaFold structure prediction of CFAST-linker-Halo displayed on a membrane bilayer. The size of Halo tag and length of flexible and helical linkers are indicated.