PIWIL4 is overexpressed in most patients with AML and AML LSCs. (A) PIWIL4 messenger RNA (mRNA) expression in types of cancer from The Cancer Genome Atlas (TCGA) data set. The boxplot indicates median expression and range of expression.²⁶ (B) qRT-PCR mRNA expression of PIWIL4 in patients with AML, bone marrow mononuclear cells (BMNCs) and CD34⁺ BM and cord blood CD34⁺ (CB CD34⁺) HSPCs. Dots indicate biological replicates; horizontal bars represent median expression. (C) mRNA expression of PIWIL4 in patients with AML vs healthy CD34⁺ BM HSPCs from the Vizome RNA-seq data set.²⁷ (D) Upper panel: PIWIL4 protein levels vs β-actin as loading control in independent biological replicates of cytogenetically normal (CN) patients with AML and healthy bulk CB. Lower panel: densitometry analysis of PIWIL4 protein expression relative to β-actin protein levels, analyzed using ImageJ. (E) RNA-seq mRNA expression PIWIL4 in de novo AML, secondary AML (sAML), and MDS. Dots indicate individual patients and the boxplot indicates median expression and range of expression. (F) PIWIL4 expression in lymphoid-myeloid primed multipotent progenitors (H-LMPPs vs L-LMPPs) derived from functionally validated, healthy individuals vs patients with leukemia patient. (G) Localization of PIWIL4 protein in AML cell line, NB-4. Nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) and nucleolus with fibrillarin. The size bar indicates 20 μm distance. (H) Fold increase in Piwil4 mRNA expression in murine HSPCs transduced with AML-specific oncogenes (MLL-AF9, Cdx2, and AML1-ETO9A) vs empty vector control, 72 hours after transduction. “n” indicates number of independent experiments.