TAG synthesis on ER is required for Rab18-controled LD growth. (A and B) Inhibition of DGAT1 activity reduced the size and number of LDs in both control (NC) and Rab18 KO cells. 1 µM of DGAT1 inhibitor (DGAT1 in.) was used. Green, LDs. Bars: 10 µm; (insets) 2 µm. Histogram in B showing the mean number of LDs in each diameter in A. Mean ± SD; n = 11–18 for each genotype; *, P < 0.05; ***, P < 0.001; NS, no significance by two-tailed t test. (C and D) Rab18 KO cells expressing DGAT1 did not accumulate mature LDs. Red, LDs. Bars: 5 µm; (insets) 2 µm. Histogram in D showing the mean number of LDs in each diameter in C. Mean ± SD; n = 19–28 cells for each genotype; *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, no significance by two-tailed t test. (E and F) Inhibition of DGAT2 did not affect the numbers and sizes of LDs in Rab18 KO cells. 2 µM of DGAT2 inhibitor was used. Bars: 10 µm; (insets) 2 µm. Histogram in F showing the mean number of LDs in each diameter in E. Mean ± SD; n = 9–11 for each genotype; *, P < 0.05; NS, no significance by two-tailed t test. (G and H) Knocking down GPAT3/4 reduced the number and sizes of mature LDs in Rab18 KO cells. Bars: 5 µm; (insets) 2 µm. Histogram in H showing the mean number of LDs in each diameter in G. Mean ± SD; n = 10; *, P < 0.05; ***, P < 0.001; NS, no significance by two-tailed t test. (I) Reduced TAG and increased ER stress in Rab18 KO cells treated with FAs. Left: Representative Western blot showing the ER stress in Rab18 KO cells. From three independent experiments. Right: Subcellular TAG levels of one representative experiment. All experiments were performed at least twice.