Aberrant expression of PIWIL4 is required for sustenance of AML growth. (A) Percentage difference in clonogenicity in CFU assay of PIWIL4-depleted vs control AML cell lines. Bars indicate mean and standard error of the mean (SEM). “n” indicates number of independent experiments. (B) Number of colonies in CFU assay of AML cell lines transduced with sgScr vs sgRNAs against PIWIL4 (sgRNA-A and B). Bars indicate mean colony number and SEM. “n” indicates number of independent experiments. (C) Flow cytometry analysis of human engraftment in BM of NSG mice that underwent transplantation with cells from patients with AML transduced with shRNA or scrambled control. Engraftment was determined by CD45 and GFP double positivity. Dots indicate engraftment levels in individual mice. (D) Flow cytometry analysis of human engraftment in BM of NSG mice that underwent transplantation with OCI-AML3 cells transduced with sgPIWIL4 or sgSCR control. Engraftment was determined by positivity for CD45. Dots indicate engraftment levels in individual mice. (E) Kaplan Meier plot showing the survival of NSG mice that underwent transplantation with PIWIL4-depleted AML cell lines. (F) Kaplan Meier plot of mice that underwent transplantation with cell number dilutions (10⁵, 10⁴, and 10³) of ckit⁺ MLL-AF9⁺ cells from primary leukemic mice transduced with shRNA against Piwil4 or scrambled control. “n” indicates the number of individual mice that underwent transplantation. The table shows calculated LSC frequencies and the right panel shows hematoxilin and eosin-stained histopathology and myeloperoxidase (MPO) immunohistochemistry of representative mice. (G) Apoptosis assay of PIWIL4-depleted cells from patients with primary AML. “n” indicates the number of biological replicates.