Figure 2. Epsin facilitates CD36-mediated lipid uptake by promoting CD36 endocytosis and recycling.

(A-C) Thioglycolate (TG) induced peritoneal macrophages from WT/ApoE^−/− (n=5) and LysM-DKO/ApoE^−/− (n=5) mice on normal diet (ND) were incubated in lipid-deficient medium for 24h and treated with or without 100μg/mL oxLDL for 1h at 37⁰C. qRT-PCR analysis of CD36 expression (A), western blot (WB) analysis for total protein level of CD36 (B) and flow cytometry for surface level of CD36 (C). (D-E) Elicited TG-induced peritoneal macrophages from WT/ApoE^−/− and LysM-DKO/ApoE^−/− mice on ND were incubated in lipid-deficient medium for 24h and treated with or without 100μg/mL oxLDL for 15min (D) or 30min (E) at 37⁰C. Macrophages were co-stained with CD36 (green), the early endosome marker EEA1 (red) or the recycling endosome marker Rab11 (Red) and DAPI (bule), and imaged using confocal microscope. White arrows indicate the endocytic vesicles, scale bar=5μm, n=8/group. (F) BODIPY staining of peritoneal macrophages from WT and LysM-DKO mice on normal diet were pre-incubated with 25μg/mL oxLDL for 24h in lipid-deficient medium, n=6/group, scale bar=10μm. (G) Cholesterol and triglycerides levels in WT and LysM-DKO macrophages treated with 25μg/mL oxLDL for 24h in lipid-deficient medium (n=6). (H) Peritoneal macrophages isolated from WT and LysM-DKO mice on normal diet were incubated in lipid-deficient medium for 24h followed by the treatment with DiI-oxLDL for 2h at 37⁰C to assess the lipoprotein uptake by flow cytometry, n=6/group. Data from A-H are presented as mean ± SD. Mann-Whitney U test was utilized in A-C. Two-way ANOVA followed by Sidak post hoc multiple comparisons test was conducted in D-E and G. Unpaired t test was conducted in F and H.