Figure 6. The atheroprotective effect of macrophage-specific TFEB overexpression is also p62-dependent.

(a) Cohorts of control and mφTFEB-TG mice (all on p62-KO and ApoE-KO background) were fed a western diet for 2 months to develop lesions—exact genotypes are provided at the top of the graph. Atherosclerotic plaque burden was quantified by computer image analysis of Oil Red O-stained aortic root sections with representative Oil Red O-stained aortic roots shown on right (scale bar, 0.4 mm; statistical significance of differences was calculated using Mann–Whitney U-test). (b) Macrophage content in aortic root sections was analysed by immunofluorescence staining using an antibody against MOMA-2 (n≥11 mice per group). (c) Apoptotic, necrotic core and combined apoptotic–necrotic core areas of aortic root sections were determined by quantification of TUNEL immunofluorescence staining, acellular areas or a combination of the two, respectively (n≥11 mice per group). (d) Immunofluorescence images of p62-KO and p62-KO/TFEB-TG macrophages treated with or without cholesterol crystals for 24 h and stained using DAPI (nuclei), and antibodies against polyubiquitinated proteins (FK-1) and p62 (scale bar, 5 μm). (e) Quantification of average ubiquitin intensity per cell from immunofluorescence experiment described in d (numbers of quantified cells are shown under each bar). (f) p62-KO and p62-KO/TFEB-TG macrophages were incubated with cholesterol crystals and the per cent of caspase 3/7-positive cells was quantified in three independent experiments (numbers of quantified cells are shown under each bar). (g) p62-KO and p62-KO/TFEB-TG macrophages were treated with LPS+CC for 24 h or with LPS followed by ATP for 3 h. Culture media were assayed for IL-1β by ELISA (n=3 independent wells for each treatment). For all graphs, data are presented as mean±s.e.m. *P<0.05, ***P<0.001, NS: not significant, two-tailed unpaired t-test, except a.