Fig. 7
Influence of the sterol content of acceptor membranes on PS delivery. a Initial rates of 16:0/18:1-PS transfer measured with Osh6p (200 nM) at 30 °C from LA (5% PS) to LB liposomes with different bulk lipid compositions and containing or not 5% 16:0/18:1-PI(4)P (at the expense of PC). Data are represented as mean ± s.e.m. (non-exchange condition, n = 4–5, exchange condition, n = 4). b Initial rates of 18:0/18:1-PS transfer from LA to LB liposomes without or with 30% or 50% cholesterol, and containing or not 5% 16:0/18:1-PI(4)P. Data are represented as mean ± s.e.m. (n = 3–6). c Experiments are similar to those shown in Fig. S9a except that 16:0/18:1-PS was replaced by 18:0/18:1-PS in LA liposomes. LB liposomes contained 0, 30, or 50% cholesterol. Each trace is the mean ± s.e.m. of kinetics recorded in independent experiments (n = 3–6). d Confocal images of live GFP-C2Lact-expressing HeLa cells (green) treated or not with U18666A for 24 h at 37 °C. Cholesterol in the PM was detected by incubating the cells for 10 min with mCherry-D4 (purple) at room temperature and then washed with medium prior to imaging. The overlay panel shows merged green and magenta images. The top-right image corresponds to a higher magnification (× 5.2) image of the area outlined in white. Scale bars: 10 μm. e Quantification of the ratio of GFP-C2Lact signal at the PM to the cytosolic GFP-C2Lact signal, as assessed by wide-field microscopy and line scan analysis. GFP-C2Lact-expressing HeLa cells with or without U18666A treatment (2.5 μg/mL for 24 h at 37 °C) as shown in d (mean ± s.e.m., n = 68 cells for non-treated cells and n = 51 cells for treated cells; data are pooled from four independent experiments for each condition). Control experiments were done with GFP-expressing HeLa cells (mean ± s.e.m., n = 37 cells for non-treated cells and n = 33 cells for treated cells; data are pooled from two independent experiments for each condition)