Figure 8. Distribution of distinct pools of cholesterol in the macrophage plasma membrane.

Mouse peritoneal macrophages were plated onto poly-D-lysine–coated MatTek dishes and incubated in medium containing 10% FBS. On the next day, cells were incubated with [¹⁵N]ALO-D4 and [¹³C]OlyA (20 μg/ml each) and then imaged by NanoSIMS. (A) Distributions of [¹⁵N]ALO-D4 and [¹³C]OlyA binding to the plasma membrane. ¹³C/¹²C NanoSIMS images revealed a broad band of ¹³C enrichment near the outer edge of the cell (corresponding to the flat lamellipodia of cells, apparent in the ¹²C^–, ¹²C¹⁴N^–, and ³²S^– NanoSIMS images). The ¹⁵N/¹⁴N images revealed a thin band of ¹⁵N enrichment at the far outer edge of the cells and in the lawn of particles on the surrounding substrate. Additional NanoSIMS images of these cells are shown (with different scales) in Figure 7 and Figure 7—figure supplement 1. (B) Composite ¹²C¹⁴N^– (blue), ¹³C/¹²C (green), and ¹⁵N/¹⁴N (red) NanoSIMS images, revealing ¹⁵N enrichment at the outer edge of the plasma membrane and in the lawn of particles on the surrounding substrate. Of note, the ¹⁵N enrichment at the outer edge of the cell extended beyond the thick band of ¹³C enrichment. In this figure, the scales for the ¹³C/¹²C and ¹⁵N/¹⁴N NanoSIMS images were adjusted for optimal visualization of the lamellipodia of macrophages. Also, ¹²C^– and ³²S^– images were included to visualize the lamellipodia. Scale bar, 5 μm. Additional NanoSIMS images of the cell in the top row of this panel are shown in Figure 8A and Figure 7—figure supplement 1; additional NanoSIMS images of the cell in the bottom row of this panel is shown in Figure 7—figure supplement 2. Two independent experiments were performed; representative images are shown. (C) Line scans comparing the ¹⁵N/¹⁴N and ¹³C/¹²C isotope ratios over the outer edge of the plasma membrane (white line in the upper and lower right images of panel B). (D) Quantification, by NanoSIMS, of [¹⁵N]ALO-D4 and [¹³C]OlyA binding to macrophages and to the surrounding particles on the substrate. ¹⁵N/¹⁴N ratios were quantified for the cell body, macrophage-derived particles, and the thin line of ¹⁵N enrichment at the edge of the cell. ¹³C/¹²C ratios were quantified for the cell body, macrophage particles, and the broad lamellipodia near the edge of the plasma membrane. For each category, twenty-five regions, 20 pixels in diameter areas were circled on the ¹²C¹⁴N^– images, and the ¹⁵N/¹⁴N and ¹³C/¹²C ratios in each circle were calculated. A minimum of six macrophage images were used. Graph shows the mean and standard deviation of the fold change of ¹⁵N or ¹³C enrichment, normalized to the macrophage cell body. **p<0.001.

Figure 8—figure supplement 1. Reduced efflux of [³H]cholesterol from [³H]cholesterol-loaded macrophages when the release of particles was blocked with blebbistatin.

Mouse peritoneal macrophages were loaded with [³H]cholesterol (1 μCi/ml) and acLDL (20 μg/ml) in macrophage growth media containing 1% lipoprotein-deficient serum (LPDS) for 24 hr. On the next day, the cells were washed and the cells were released with 5 mM EDTA. The cells were then re-plated in six-well plates (1 × 10⁶ cells/well) in medium containing blebbistatin (30 μM), an LXR agonist (1 μM), or vehicle (DMSO) alone. After 24 hr, the macrophages were washed, and both the macrophages and the particles were released from the plates by incubating the cells with 5 mM EDTA. (The complete release of particles and cells from the substrate was documented by SEM). Radioactivity in macrophages (A) and particles (B) was measured by scintillation counting. Graph shows the mean and standard deviation of radioactive counts normalized to the protein content from three independent experiments. **p<0.001 or *p<0.01 compared to cells incubated with DMSO alone.

Figure 8—figure supplement 2. Increased efflux of [³H]cholesterol from [³H]cholesterol-loaded macrophages in the presence of HDL.

Mouse peritoneal macrophages were loaded with [³H]cholesterol (1 μCi/ml) and acLDL (20 μg/ml) in macrophage growth media containing 1% LPDS for 24 hr. On the next day, cells were washed, released from the plate with 5 mM EDTA, and then re-plated on a 6-well plate (1 × 10⁶ cells/well) in serum-free macrophage growth medium containing blebbistatin (30 μM), an LXR agonist (1 μM), or vehicle (DMSO) alone. In some samples, HDL (20 μg/ml) was included in the medium. Cells were grown for 24 hr and then released with EDTA. The cell culture media was removed, centrifuged to remove cellular debris, and the radioactivity in the cell culture medium was measured by scintillation counting. Graph shows the mean and standard deviation of radioactive counts normalized to the protein content from two independent experiments. **p<0.001.