Impaired mitophagy in leukemia cells after loss of p62. (A) shCtrl and shp62 MN1-driven ldMBM leukemia cells were treated for 12 hours with 1 mM DFP. Lysates were analyzed by western blotting for the inner mitochondrial protein COXIV and the outer mitochondrial protein Tom20 and the autophagic markers p62 and LC3. β-Actin served as a loading control. (B) Quantification of remaining protein expression after 12 hours of treatment with DFP of mitochondrial proteins Tom20 and COXIV compared with 0 hours using density analysis of western blots (n = 3 independent experiments). (C) Immunofluorescent staining of shCtrl and shp62 MN1-driven ldMBM leukemia cells after 12 hours of treatment with 1 mM DFP was performed with the mitochondrial and autophagic markers Tom20 and LC3, respectively. Arrows indicate colocalization of both markers. Tom20 and LC3 were immunostained with fluorescent dyes Alexa Fluor 647 and Alexa Fluor 593, respectively. DAPI (4′,6-diamidino-2-phenylindole) was used as a nuclear stain. Scale bar = 5 µm. Original magnification ×63. (D) Colocalization of Tom20 and LC3 was analyzed by ImageJ (n = 50 for each group). (E) Immunofluorescent staining of shCtrl and shp62 MN1-driven ldMBM leukemia cells after 12 hours of treatment with 1 mM DFP was performed with the mitochondrial and lysosomal markers Tim23 and Lamp1, respectively. Arrows indicate colocalization of both markers. Tim23 and Lamp1 were immunostained with fluorescent dyes Alexa Fluor 647 and Alexa Fluor 593, respectively. SYTOX green was used as a nuclear stain. Scale bar = 5 µm. Original magnification ×63. (F) Colocalization of Tom20 and Lamp1 was analyzed by ImageJ (n = 50 for each group). Values are means ± SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001.