p62 levels correlate with survival, and p62 depletion impairs cell growth in human and murine leukemia cells. (A) Kaplan-Meier survival curves of adult AML patients were stratified by lowest (red) and highest (black) p62 gene expression (n = 40 in lowest quartile, n = 41 in highest quartile; P = .026), which was analyzed from RNA sequencing data and survival data from the TCGA LAML data set containing 173 adult patients with de novo AML. (B) Competitive cell growth of CRISPR/Cas9 targeted human p62 (Crp62, GFP⁺) and human nontargeting control (CrNTC, GFP⁻) in human AML cell lines THP1, U937, NB4, and Molm13 measured by flow cytometry on indicated days (pooled data from 3 independent experiments). (C) Quantification of cell proliferation of shp62 and scrambled nucleotide control short hairpin RNA (shCtrl) MN1- and HoxA9/Meis1-driven ldMBM leukemia cells was performed by seeding 10⁴ cells and counting live cells (by Trypan blue exclusion) at the indicated time points. (D) Colony numbers and (E) cell numbers were determined in serial colony-forming unit assays of p62 knockdown (shp62) and control (shCtrl) MN1-driven ldMBM leukemia cells. (F) shp62 and shCtrl MN1-driven ldMBM leukemia cells were stained with BrdU and 7AAD and analyzed by flow cytometry. The graph shows percentage of cells in each cell-cycle phase (G0/G1, BrdU⁻ 2 N DNA content; S: BrdU⁺; G2/M, BrdU⁻ 4N DNA content). All experiments in panels B-F were performed in triplicate. The statistical significance between 2 groups was analyzed by a standard Student t test. Error bars represent SEM. *P ≤ .05; **P ≤ .01; ***P ≤ .001.