Figure 1.

Detection of chaperone-mediated autophagy marker LAMP-2A (lysosome-associated membrane protein type 2A) in vivo and in vitro. A, Representative Western blot analysis of LAMP-2A in mouse primary peritoneal macrophages (MØ), primary aortic smooth muscle cells (SMCs), and primary aortic endothelial cells (ECs). B, Representative immunohistochemical images for detecting LAMP-2A, MOMA-2 (specific for monocytes and macrophages), and α-SMA (smooth muscle actin; specific for SMCs) in three consecutive frozen sections from the aortic root of ApoE^−/− mice (male) fed a high-fat diet (HFD) for 8 wk. n=5 per group. The positive reactions of tissue sections were displayed as red color. Scale bar=100 μm. C, Representative immunofluorescence analysis for detecting LAMP-2A (green particles), MOMA-2 (red particles), and α-SMA (red particles) in frozen sections from the aortic root of ApoE^−/− mice (male) fed a HFD for 8 wk. n=5 per group. Scale bar=100 μm. D, Representative immunofluorescence analysis for detecting the colocalization (yellow particles) of LAMP-2A (green particles) and CD68 (specific for monocyte macrophages, red particles) in frozen sections from human coronary atherosclerotic plaques. Scale bar=100 μm. E, Representative Western blot images and (F–H) quantitative analysis of protein expression of PLIN-2 (perilipin-2), LAMP-2A, and LAMP-1 of mouse peritoneal macrophages from wild-type (WT) and L2A-mØKO mice (male) treated with or without ox-LDL (oxidized low-density lipoprotein; 40 and 80 μg/mL) for 24 h. Five independent experiments were performed. Data were presented as medians and quartiles. ox-LDL 80 μg/mL group and ox-LDL 40 μg/mL group were compared with PBS group respectively. Statistical analysis was conducted using Kruskal-Wallis 1-way ANOVA with Nemenyi post hoc test.