Inhibition of Ser synthesis causes cellular stress in leukemic cells (A) Cell cycle distribution of normal and leukemic HSPCs after a 48-hour block of Ser biosynthesis. Shown are means and standard deviations of 3 independent experiments. Cell cycle phases are colored as indicated. (B) Cellular ROS in cells treated as in panel A. The cells were stained with CellROX deep red reagent and evaluated by FACS. A representative example of 2 experiments is depicted. (C) DNA damage after Ser synthesis blockade. Nuclear proteins were extracted from cells treated as before and immunoblotted with antibodies against the damage-specific histone derivative γ-H2AX and for histone H3 as the control. (D) ROS quenching restores CFC activity after CBR5884 treatment. MLL-ENL–transformed cells were incubated for 48 hours with 8 µM CBR5884 and either solvent or the indicated concentrations of the ROS-quencher NAC. The number of CFCs was recorded in triplicate experiments, with results depicted as mean and standard deviation. An example of the CFC assay is shown in the right panel. DMSO (left columns) difference between NAC 0 mM to 0.4 mM is not significant; difference between 0 mM and 1 mM NAC is P = .03. CBR5884 (right columns) difference between 0 mM and 0.4 mM NAC is P = .00002; difference between 0 mM and 1 mM NAC is P = .002. (E) Steady state levels of p53 in normal and leukemic HSPCs, as detected by western blot. (F) Blunted p53 response in leukemic cells. Expression levels of the major p53 target genes Cdkn1a/p21 and Mdm2, as well as those of the p53 antagonist Myc were determined by qPCR in normal and leukemic HSPCs, in steady state and after CBR5884 treatment.