Figure 2

NSlit2 inhibits uptake of oxLDL by macrophages. (a), M-CSF-induced human macrophages were incubated with vehicle, NSlit2 or CSlit2, then incubated with oxLDL for 24 h. Internalized oxLDL was labeled with BODIPY. Representative images were obtained using a spinning disk confocal microscope at 63× magnification. Cell borders are indicated by dashed lines. Scale bar, 10 μm. (b), Experiments were performed as in (a) using GM-CSF-induced human macrophages. (c), Experiments were performed as in (a). Cells were then detached and BODIPY mean fluorescence intensity (MFI) was measured by flow cytometry. (d), Experiments were performed as in (b). Cells were then detached and BODIPY MFI was measured by flow cytometry. (e), M-CSF-induced human macrophages were preincubated with vehicle, NSlit2 or Robo1N + NSlit2 followed by incubation with oxLDL for 2 h. Internalized oxLDL was labeled with BODIPY. Representative images using a spinning disk confocal microscope at 40× magnification. Cell borders are indicated via dashed lines. Scale bar, 10 μm. (f), Quantification of experiments performed in (e). Individual dots correspond to single cells. Mean ± SEM from 4 independent experiments. (g), M-CSF-induced human macrophages were incubated with vehicle, NSlit2 or CSlit2, followed by incubation with DiI-oxLDL for 20 min. Cells were washed, detached and internalized DiI-oxLDL MFI was measured by flow cytometry. Data are the mean ± SEM of 8 independent experiments. (h), Experiments were performed as in (g) using GM-CSF-induced macrophages. Data are the mean ± SEM of 3 to 5 independent experiments. For (c), (d), samples were acquired using a Gallios flow cytometer (Beckman Coulter-US) with Kaluza Acquisition software (Beckman Coulter-US) and analyzed with FlowJo v10 (BD Biosciences-US). For (c), (d), (f)–(h), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were determined by one-way ANOVA using Tukey’s post hoc test.