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Figure 4. VSMCs of the aortic media and cultured aortic VSMCs of old age LCR rats show impaired mitophagy. Representative confocal images of aortic cross sections (A, top panel) were co-stained for LAMP1 (green), TOM20 (red) and SM-α-actin (blue). The white fluorescence indicates colocalization of all proteins and active mitophagy in medial VSMCs. VSMCs (B, top panel) were co-stained for LAMP1 (green) and TOM20 (red). Bright yellow fluorescence indicates colocalization of LAMP1 and TOM20 and active mitophagy. Data are presented as mean integrated density/ROI ± SEM where n = 4 independent cell lines (A and B, bottom panels). Representative confocal images of mitophagy flux, showing bright yellow colocalization of Mtphagy-(red) and Lyso-(green) dyes (C, top panel; scale 10 μm). Data are mean integrated density/ROI ± SEM. Representative images of autophagic vacuole formation were measured by the green fluorescence puncta of a specific dye, CYTO-ID (ENZO) (D, top panel; scale 10 μm). Data are the average number of punctate vacuolar structures per cell (number) or the average pixel area of fluorescence integrated density/ROI ± SEM (D, bottom panel). Western blot analysis and densitometric quantification of mitophagy/autophagy marker LC3B in VSMC lysates (E). Data are mean ± SEM where n = 4. Representative immunohistochemistry images of VSMC stained with Sudan Black B (F, top panel; scale = 50 μm). Data are presented as mean integrated density/ROI ± SEM where n = 4 (F, bottom panel). *P < 0.05; **P < 0.01; ***P < 0.001.