Effect of the enhancer mutation on HL-60 cell differentiation during growth or after PMA stimulation. (A) Comparison of neutrophil and monocyte markers on unstimulated HL-60 wt and A-to-T enhancer mutant cell lines after passage for 1 or 3 days. Cells were surface stained with an anti-human CD14 monoclonal antibody for monocyte differentiation (X-axis) and with an anti-human CD66b monoclonal antibody for neutrophil differentiation (Y-axis). Boxes indicate cells that are CD14^– CD66⁺ (neutrophils) or CD14⁺ CD66b ^–/+ (monocytes or less differentiated cells). (B) Relative GATA2 RNA levels were measured +/− PMA by dPCR or RNA-seq: wt change 0.77 to 4.44, mutant change 0.92 to 1.47. Levels of GATA2 RNA were normalized with TBP (dPCR) or fragments per kilobase of transcript per million mapped reads (RNA-seq). (C) Cells were surface stained for CD11b (Y-axis) and intracellularly stained for GATA2 (X-axis) for control (CTL) or PMA-stimulated cells. All cells expressed GATA2 and were gated into low or high expressing cells (right quadrants), with the percentages for each quadrant shown. The mean florescence intensity (MFI) values are given.