Reintroduction of WT p62 can rescue the phenotype, but not the ΔLIR mutant of p62. (A) Expansion analysis of shCtrl (+ mock), shp62 (+ mock), and shp62 (+ p62 WT, p62 ΔLIR, and p62 K7A/D69A) transduced MN1-driven ldMBM leukemia cells were performed by seeding 10⁴ cells and counting live cells (by Trypan blue exclusion) at the indicated time points (n = 3 in triplicate). (B) Colony numbers and (C) cell numbers were determined in colony-forming unit assays of shCtrl, shp62, and p62 mutants transduced MN1-driven leukemia cells (n = 3 in triplicate, each with 500 cells). (D) shCtrl, shp62, and p62 mutants transduced MN1-driven leukemia cells were treated for 12 hours with 1 mM DFP. Lysates were analyzed by western blotting for the inner mitochondrial protein COXIV and p62. β-Actin served as a loading control. (E) Quantification of remaining protein expression after 12 hours of treatment with DFP of mitochondrial protein COXIV compared with 0 hours using density analysis of western blots (n = 3 independent experiments). (F) Colocalization of mitochondrial (Tom20) and autophagic (LC3) marker after treatment with 1 mM DFP in shCtrl, shp62, and p62 mutants transduced MN1-driven leukemia cells was visualized by immunofluorescent staining. 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 counterstain. Scale bar = 5 µm. Original magnification ×63. (G) Colocalization of Tom20 and LC3 was analyzed by ImageJ (n = 50 for each group). Values are means ± SEM. ns, not significant; *P ≤ .05; **P ≤ .01; ***P ≤ .001.