Figure 1. Atherogenic lipid loading and autophagy deficiency lead to p62-enriched polyubiquitinated protein accumulation in macrophages.

(a to c) Western blot analysis and (c) immunofluorescence of p62 in peritoneal macrophages after cholesterol crystal (CC) or oxidized LDL (oxLDL) incubation for the indicated times. β-actin was used for loading control and densitometric quantification from three separate experiments is shown next to the representative western blot (a,b). Nuclei were counter stained with DAPI and graph represents average p62 dot numbers per cell after the indicated treatments (n≥35 cells for each group in three independent experiments; c, right). (d–e) Immunofluorescence images of control and ATG5-KO peritoneal macropahges after cholesterol crystal incubation for the indicated times using DAPI and antibodies against ubiquitinated proteins (FK-1) and p62. (f) Graphs represent average p62/ubiquitin+ dot numbers (top) and average ubiquitin intensity (bottom) per cell for immunofluorescence images in d, e (n≥30 cells for each group in three independent experiments, #P<0.05 compared to the respective time-point in Control cells). (g) Western blot analysis of polyubiquitinated proteins (FK-1 antibody) in detergent soluble and insoluble lysate fractions of cholesterol crystal-treated control and mΦATG5^−/− peritoneal macrophages. Densitometric quantification from three separate experiments is shown to the right. (h–j) Immunofluorescence images of control and mΦATG5^−/− peritoneal macrophages after cholesterol crystal incubation for the indicated times using antibodies against the late endosome/lysosome marker LAMP2 and p62. Graph represents percent of p62+ dots co-localizing with LAMP2 (n≥15 cells for each group in three independent experiments; j). For all panels, data are presented as mean ± SEM. (*P<0.05, **P<0.01, ***P<0.001, NS = not significant). Scale bar, 5 μm.