FIGURE 6:

Acute free cholesterol accumulation induces association of ApoB with LDs. (A) F1394,10 μg/ml, was added to Huh7 cells. At appropriate intervals, cells were fixed, permeabilized with digitonin, and doubly labeled with BODIPY 493/503 and anti-ApoB antibody as described in Materials and Methods. Bar, 10 μm. (B) Cells were treated with MβCD/cholesterol in LPDS (Chol) or MβCD/cholesterol plus 58-035 in LPDS (Chol + 58-035) as described in Figure 1. Cells were then fixed, and ApoB was labeled as described. (C) Huh7 cells were cultured for 24 h in the absence (Control) or presence of 10 μg/ml F1394. Cells were then homogenized, and PNS was separated by stepwise sucrose density gradient as described in Materials and Methods. The distribution of ApoB was measured by Western blotting. (D) Huh7 cells were grown for 24 h in the absence and presence of 10 μg/ml F1394. Cells were then fixed and permeabilized with 0.1% Triton X-100. Intracellular distribution of ApoB and PDI was examined as described in Materials and Methods. Bar, 10 μm. (E) Cells were treated as described and then fixed and permeabilized with 50 μg/ml digitonin, followed by double labeling with anti-ApoB and anti-PDI. Bar, 10 μm. (F) Cells were incubated as in A, followed by labeling with ER tracker. Bar, 10 μm. (G) Cells were treated as in D and fixed and permeabilized with 50 μg/ml digitonin, followed by double labeling with anti-PLIN2 and anti-ApoB. Bar, 10 μm.