(A) Analysis of intracellular distribution fluorescently labeled PC following administration of ER-targeting liposomes. Fluorescently labeled ER-targeting liposomes enriched for different PC species were formulated as described in Methods. The PE, PS, and PI components were identical between liposomes, and the PC component was contributed by defined species as indicated. Liposomes were labeled with 1% bodipy-conjugated PC (green). DAPI (blue, left panels) and ER tracker (red, right panels) were used for costaining. Scale bars: 20 μm. (B) Quantification of ER-targeting liposome uptake. Cells were treated with fluorescently labeled carrier liposome enriched for either 18:0/18:0 PC or 18:0/20:4 PC for 1 hour. Cells were washed with PBS and lysed in RIPA buffer and fluorescence was measured with a plate reader. (C) Primary hepatocytes from C57BL/6 mice were infected with adenovirus expressing HSV–tagged SREBP-1c for 24 hours and treated with the indicated ER-targeting liposome (30 μM total phospholipid concentration) for the last 3.5 hours. Membrane and nuclear fractions were isolated and analyzed by immunoblotting. (D) Quantification of Western blot results from 3 independent experiments shown in part C. *P < 0.05, Student’s t test. (E) Primary hepatocytes from C57BL/6 mice were infected with adenovirus expressing HSV-tagged SREBP-1c for 24 hours and treated with liposomes composed of the indicated single PC species at 30 μM for the last 3.5 hours. Membrane and nuclear fractions were isolated and analyzed by immunoblotting. (F) Primary hepatocytes from Scap^fl/fl mice were infected with adenoviral GFP or Cre recombinase and treated with GW3965 (1 μM) for 24 hours. Protein from total cell lysates was analyzed by immunoblotting.