Figure 7: ANGPTL4 deficiency promotes macrophage foam cell formation and apoptosis.

(a) Representative pictures from WT and Angptl4^−/− mouse peritoneal macrophages incubated with or without Ac- LDL (120 μg ml⁻¹) for 24 h and stained with BODIPY 493/503 (1 μg ml⁻¹) and DAPI (Green and blue, respectively). Scale bar, 5 μm. Quantification of the mean average intensity is in the right panel. (b) Total cholesterol content in peritoneal macrophages isolated from WT and Angptl4^−/− mice incubated with or without Ac-LDL (120 μg ml⁻¹) for 24 h. (c) Flow cytometry analysis of DiI-Ox-LDL binding in peritoneal macrophages incubated with DiI-Ox-LDL (30 μg cholesterol per ml) for 30 min at 4 °C. At the end of the incubation period, cells were washed and incubated in RPMI 10% FBS media for 15 min at 37 °C to allow the internalization. (d) Flow cytometry analysis of DiI-Ox-LDL uptake in peritoneal macrophages incubated with DiI-Ox-LDL (30 μg cholesterol per ml) for 2 h at 37 °C. The results are expressed in terms of specific MFI after subtracting auto-fluorescence of cells incubated in the absence of DiI-Ox-LDL. (e) Cholesterol efflux to apolipoprotein A1 (ApoA1) in peritoneal macrophages isolated from WT and Angptl4^−/− mice stimulated with or without T0901317 (T090). (f) Western blot analysis of indicated proteins in peritoneal macrophages from WT and Angptl4^−/− mice incubated with or without Ac-LDL (120 μg ml⁻¹) for 24 h. (g) Western blot analysis (representative of three blots) of ABCA1 expression in WT and Angptl4^−/− peritoneal macrophages incubated with Ac-LDL for 24 h. Surface ABCA1 was isolated using biotinylation followed by incubation with neutravidin. HSP90 is used as loading control (f and g). Full scans of westerns blots are provided in Supplementary Fig. 8. (h) Representative confocal images of mouse peritoneal macrophages from WT and Angptl4^−/− mice incubated with Ac-LDL for 24 h and stained with cholera toxin B (CTxB), ABCA1 and DAPI. Quantification of co-localization of CTxB and ABCA1 is on the right panel. Scale bar, 10 μm. (i) Representative images of WT and Angptl4^−/− macrophages cultured on coverslips and treated with or without Ac-LDL (120 μg ml⁻¹) in combination with ACAT inhibitor (58035) for 24 h to induce lipid-loading-induced apoptosis (scale bars, 200 μm). Apoptosis was detected using Annexin-V staining. Right panel shows the quantification of percentage of apoptotic cells from four random fields from each cover slip. All data represent the mean±s.e.m. from at least three experiments in duplicate; *P<0.05 compared with WT macrophages by unpaired t-test. MFI, median intensity of fluorescence.