ER-associated SNAREs control LD growth by interacting with Rab18/NRZ. (A–D) KO ER-associated Q-SNAREs (Stx18, Use1, and BNIP1) in 3T3-L1 preadipocytes led to defective LD growth and the accumulation of supersized LDs. (A) Western Blot showing the expression level of indicated protein in 3T3-L1 preadipocytes. (B) Representative images of LD morphology. Bars: 10 µm; (insets) 2 µm. (C) Histogram showing the number of LDs in each diameter in B. (D) Quantification of the number of mature LDs in B. Mean ± SD; n = 15–20 for each genotype; ***, P < 0.001 by one-way ANOVA with Tukey post hoc tests. (E) The size distribution of the largest LD in each cell in B. The diameters of LDs from 218–252 cells/genotype (pooled from three experiments) were fitted with Gaussian function. (F) Reduced cellular TAG levels in Stx18-deficient, Use1-deficient, or BNIP1-deficient 3T3-L1 preadipocytes. Three independent experiments were performed. Mean ± SD; n = 3; ***, P < 0.001 by one-way ANOVA with Tukey post hoc tests. (G) LD morphology in 3T3-L1 preadipocytes knocking down R-SNAREs (Sec22b, Ykt6, and Vamp8). Green, LDs. Bars: 10 µm; (insets) 2 µm. (H) The LD size distribution in G. The diameters of LDs from 97–206 cells/genotype (pooled from three experiments) were fitted with Gaussian function. (I) Rab18 interacts with ER-associated Q-SNAREs. (J and K) The LD association of ER-associated Q-SNAREs is dependent on Rab18 by biochemical fractionation (J) and imaging (K) analyses. Subcellular fractions were isolated from control or Rab18 KO TM-3 cells. In K, Q-SNAREs, LD, and Cherry-Rab18 were labeled green, blue, and red, respectively. Bars: 10 µm; (insets) 2 µm. All experiments were performed at least twice.