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Fig. 5. (A) Immunogold electron microscopy of HepG2 cells expressing EGFP-Rab18(WT). Ultrathin cryosections were labeled with anti-GFP antibody. Most LDs appeared as vacant round spaces because lipid esters were not retained well in the sections. Gold labeling was observed along the rim of LDs. Notably, thin membrane cisternae were often seen adjacent to the labeled LD (arrows). Scale bar: 100 nm. (B) Conventional electron microscopy of 3T3 cells expressing EGFP-Rab18(WT). LDs were frequently apposed to thin membrane cisternae (arrows). The direct continuity of the membrane cisterna and the rough ER (double arrows in the upper left, upper right and middle right figures), and the ribosomes in the membrane cisterna (double arrows in the lower right figure) were observed in many cases. Scale bars: 100 nm. (C) Conventional electron microscopy of control 3T3 cells. Small ER cisternae were occasionally found adjacent to LDs (arrowheads), but contacts between them were seldom extensive. Scale bar: 100 nm. (D) 3T3 cells treated with siRNA for knockdown of the expression of ADRP. (D-1) Western blotting of ADRP. Cells were treated with control random siRNA or ADRP siRNA, and equal amounts of the total cell lysates (20 µg) were electrophoresed. The expression of ADRP was reduced by more than 50% by this procedure. (D-2) In cells treated with ADRP siRNA, LDs in apposition with thin membrane cisternae were frequently observed (arrows). Scale bar: 100 nm. (E) Conventional electron microscopy of 3T3 cells treated with brefeldin A for 5 hours. Most LDs were surrounded by thin membrane cisternae (arrows). Scale bar: 100 nm. (F) Frequency of 3T3 cells with LDs in close membrane apposition. More than 30 cells were chosen randomly, and only LDs with membrane cisternae apposed to more than half its circumference were counted as positive. The transfection efficiency was no less than 60% as determined by fluorescence microscopy.
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