Significantly reduced HK3 and PU.1 mRNA levels in primary AML patient samples and induction of HK3 expression during neutrophil differentiation of NB4 APL cells. (A) Primary AML blasts were isolated using a Ficoll gradient, total RNA was extracted, and HK3 (left panel) and PU.1 (right panel) mRNA levels were measured by real-time quantitative RT-PCR. Measured cycle threshold (C_t) values represent log2 expression levels. Values were normalized to the expression levels of the housekeeping genes HMBS and ABL1. ***P < .0001. ‡P < .05. (B) NB4 and NB4-R2 cells were differentiated with 1μM ATRA for 6 days. HK1, HK2, and HK3 mRNA were measured by real-time quantitative RT-PCR and given as n-fold changes compared with untreated SHCOO2 cells and normalized to the housekeeping gene HMBS. ***P < .0001. (C) PU.1 and HK3 protein expression was measured by Western blotting in NB4 and NB4-R2 treated as in panel B. GAPDH expression was used as loading control. (D) CEBPE mRNA expression was determined in NB4 cells treated as in panel B. ***P < .0001. (E) CD11b flow cytometric statistical analysis of NB4 or NB4-R2 cells treated as in panel B. *P < .01 (all P values Mann-Whitney U tests).