Rab18 controls the growth of nascent LDs. (A–C) Similar number of nascent LDs labeled with GFP-LiveDrop in control and Rab18 KO cells. Green, GFP-LiveDrop; red, LipidTOX neutral red. Circles, LipidTOX-positive LDs; dotted line circles, LipidTOX-negative LDs. Bars: 5 µm; (insets) 2 µm. (B) Quantification of the number of LiveDrop-positive LDs and LipidTOX-positive LDs. Mean ± SD; n = 10 for control cells, n = 11 for Rab18 KO cells; NS, no significance; ***, P < 0.001 by two-tailed t test. (C) The percentage of LDs that were LiveDrop-positive but LipidTOX-negative. Mean ± SD; n = 10 for control cells, n = 11 for Rab18 KO cells; ***, P < 0.001 by Mann-Whitney test. (D) Representative images showing LiveDrop-positive (green) LDs in cells treated with OA for 4 h. Red, LipidTOX neutral red. Bars: 5 µm; (insets) 2 µm. (E) Representative EM images showing the LD morphology in Rab18 KO and in control 3T3-L1 preadipocytes. Red arrows, the nascent LDs; orange arrows, the mature LDs; blue arrows, the supersized LDs. Bars, 500 nm. (F) Number of LDs in randomly selected EM image fields in cells treated with OA for 1 h in E. Mean ± SD; n = 147 ROIs for control cells, n = 154 ROIs for Rab18 KO cells; ***, P < 0.001 by Mann-Whitney test. (G and H) Total number of LDs (G) and the mean number of LDs in each diameter in cells (H) that were treated with OA for 8 h in E. Mean ± SD; n = 22 ROIs for control cells, n = 29 ROIs for Rab18 KO cells; **, P < 0.01; ***, P < 0.001; NS, no significance by Mann-Whitney test. All experiments were performed at least twice.