Fig. 3. EM revealed that the ApoB-crescent is a flat ER cistern fused to an LD. (A) Pre-embedding immunoEM of Huh7 cells treated with 0.4 mM DHA. The labeling for both ApoB and PDI was localized to the thin cisternal lumen (arrowheads) adhering to the LD. ApoB was labeled by a goat anti-ApoB antibody. High magnification pictures clearly show the presence of the cisternal membrane (arrows). Bars, 500 nm. (B) Conventional EM of Huh7 cells treated with 10 µM ALLN (a,b,e) or 0.4 mM DHA (c,d,f) for 2-12 hours. (a-d) A flat membrane cistern (arrowheads) was observed along the LD perimeter. Some of the cistern was continuous with the rough ER (arrows) or decorated with ribosomes (c). The electron density of the LD is high in DHA-treated samples because osmium tetroxide reacts with unsaturated bonds of DHA. (e,f) High magnification micrographs delineated that the cisternal membrane with a unit membrane appearance (blue arrowheads) ends on the LD surface that lacks the visible membrane (green arrowheads). (g) A scheme of the membrane leaflet continuity between the cisternal membrane and the LD surface. Bars, 500 nm. (C) The ratio of cells with an LD-associated membrane cistern was quantified in EM pictures and compared with the ratio of cells with ApoB-crescents by immunofluorescence microscopy. The two ratios showed a good correlation in Huh7 cells treated with 0.4 mM DHA for 2, 6, and 12 hours. (D) The width of the LD-associated membrane cistern. Ten cisterns were randomly selected from Huh7 cell samples treated with 10 µM ALLN or 0.4 mM DHA for 12 hours. The width of each cistern was measured at four different points. (E) Direct continuity of the cisternal membrane and the LD surface was maintained in the isolated LD preparation. The cisternal membrane (arrowheads) was disrupted in most samples. Bar, 500 nm. (F) Glucose-6-phosphatase activity was visualized by enzyme histochemistry using lead nitrate. The reaction product was found in the LD-fused membrane cistern (arrowheads) and in the ER (arrows). Bar, 500 nm. (G) The ApoB-crescents in cells treated with 0.4 mM DHA for 2, 6, and 12 hours were photographed by EM, and the ratio of crescents that were directly continuous with the ER was quantified. The diameter of LD that contributed to the ApoB-crescent was also measured for the same sample and averaged.