Figure 3.

NSlit2-mediated inhibition of oxLDL uptake by macrophages is CD36- and Rac1-dependent. (a), M-CSF-induced human macrophages were incubated with anti-CD36 Ab or with an isotype-matched IgG1 Ab for 30 min, followed by incubation with vehicle, NSlit2, or CSlit2. Macrophages were then incubated with oxLDL for 24 h and labeled with BODIPY to visualize intracellular lipid droplets. Representative images were acquired using a spinning disk confocal microscope. BODIPY-labeled oxLDL puncta are shown pseudocoloured white and cell borders are indicated via dashed lines. Scale bar, 10 μm. (b), Quantification of experiments performed in (a). BODIPY MFI was quantified using ImageJ Software and 20–30 cells per experimental group were analyzed. Data are the mean ± SEM of 3 independent experiments. (c), BMDM from wild-type (WT) and CD36^−/− mice were incubated with NSlit2 at 4 °C followed by incubation with AF-647-conjugated anti-His Ab at 4 °C. Cells were fixed and representative images of cell surface labeling were acquired using a spinning disk confocal microscope. Dashed lines indicate cell borders. Scale bar, 10 μm. (d), Quantification of (c). His647 MFI was quantified using Volocity Software and 20–30 cells were analyzed for each experimental condition. Individual dots correspond to single cells pooled from 4 independent experiments. Vertical bars represent the mean ± SEM of the pooled data. Comparison was determined by an unpaired, t-test, and was not significant. (e), BMDM from WT, Rac1^−/−, and Rac2^−/− mice were incubated with vehicle, NSlit2, or CSlit2, then with oxLDL for 24 h. Internalized oxLDL was labeled with BODIPY and measured as described in (b). BODIPY MFI was measured in 40–50 cells per experimental group. Data are the mean ± SEM of 3 independent experiments. For (b) and (e), **p < 0.01, ***p < 0.001, ****p < 0.0001 were determined by one-way ANOVA using Tukey’s post hoc test.