FIGURE 8:

Cytosolic injection of MCD relieves the increase in free cholesterol observed in cells silenced for STARD4, and rescues sterol transport defects. (A) Left panel, wide-field fluorescence microscopy images of U2OS cells transfected with siSTARD4 for 48 h, then subjected to microinjection of MCD along with rhodamine-dextran, or with the tracer rhodamine-dextran alone as a control (buffer injection). Following injections, the cells were incubated for 2 h at 37°C. Cells were then fixed, stained with filipin, and imaged. The right panel shows quantification of filipin fluorescence power per microinjected cell. Fluorescence power was normalized to a control experiment in which the transfection reagent alone was added to the cells. Measurements were performed on 30 injected cells for each condition. Scale bar: 20 μm. (B) Control or STARD4-depleted cells were labeled with DHE and microinjected (or not) with MCD/rhodamine-dextran. Cells were placed in growth medium (1% LPDS) supplemented with 500 μM cholesterol complexed with MCD and 10 μM mevinolin for 2 h at 37°C. Before imaging, cells were labeled with the lipid-droplet marker LipidTOX Green. Right panel represents quantification (± SE) of three independent experiments. Measurements were performed on a total of 70 injected cells for each condition.