Methionine deprivation significantly impairs protein translation in AML and healthy HSPC. Polysomal profiling in the AML cell lines MOLM13 and HL60 along with PBMC-derived CD34⁺ cells. (A) Images of agarose gels loaded with RNA isolated from sucrose gradient input (Inp) sample (not run through gradient) and sucrose gradient− derived RNA fractions of MOLM13 cells grown for 24 and 48 hours with and without methionine. (B) Percentage of RNA in the sucrose gradients derived fractions as percentage of total RNA of all fractions in MOLM13 cells at 24 and 48 hours. (C) Percentage of polysomal and sub-polysomal RNA as a fraction of total cellular RNA in MOLM13, HL60, and PBMC CD34⁺ cells. Polysomal profiling was performed once for each cell type but in 3 experiments independent of each other. (D) Antipuromycin western blot performed on HL60 and MOLM13 cells following puromycin pulsing after 24 hours’ growth in the presence or absence of methionine. First lanes are a negative control with lysates from cells not exposed to puromycin and a cycloheximide-treated positive control (n = 3). Replicates in supplementary Figure 6E-F. β-Actin was used as loading control. (E) Relative total RNA of AML cell lines grown in the absence and presence of methionine for 24 and 48 hours. (F) Relative total protein concentration of AML cell lines grown with and without methionine for 48 hours. All experiments: error bars represent mean ± SEM. Statistical analysis by Student t test, ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001.