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. 2020 Oct 20;6:107. doi: 10.1038/s41420-020-00342-7

Fig. 1. SQSTM1/p62 constitutively delivers mitochondria to lysosomes for degradation in the absence of exogenous damage.

Fig. 1

a U2OS cells were treated with vehicle (DMSO) or with 50 nM Bafilomycin A1 for 2 h to block degradation of lysosomal contents and analyzed by fluorescence confocal microscopy. Representative images are shown. TOMM20 (Green), p62 (purple), LAMP1 (red), Dapi (blue). White arrows show instances of mitochondria tagged by p62 and surrounded by lysosomes. Yellow arrows designate mitochondria that are not being degraded. b Representative high-resolution confocal image of cells treated with bafilomycin A1 as in a. Tomm20 (green), p62 (purple), LAMP1 (red). c U2OS mito-mCherry cells were treated with 50 nM Bafilomycin A1 or vehicle (DMSO) for the indicated time. Mitochondrial accumulation was detected by flow cytometry and displayed as percent increase over time. d Protein levels of p62 from U2OS cells that were treated with the indicated concentrations of Bafilomycin A1 overnight were detected by western blotting analysis. TFIIH was used as a loading control. e U2OS mito-mCherry cells were transfected with the indicated siRNA oligos or siControl (the p62 Si pool contains oligos 1–4). Seventy-two hours later, mitochondrial accumulation was measured by flow cytometry. Mean ± SE of n = 3 independent experiments is shown; ***p < 0.001; ****p < 0.0001. f p62-knockout U2OS cells were stained with 100 nM MitoTracker Green FM for 30 min, and mitochondrial accumulation was measured by flow cytometry. Mean ± SE of n = 7 independent experiments is shown; **p < 0.01; ****p < 0.0001.