NmU rescues erythroid colony formation when endogenous c-myb expression is silenced in primary human CD34⁺ cells. (A) Primary human CD34⁺ cells were cultured with IL-3, IL-6, and SCF for 8 days and nucleofected with either full-length NmU promoter or mutant NmU promoter constructs in the absence or presence of c-myb expression construct. After 48 hours, luciferase activity was measured and presented as mean fold induction of a duplicate determination. (B) Primary human CD34⁺ cells were nucleofected with control or c-myb siRNA; and 48 hours after nucleofection, the expression of c-myb was determined by quantitative real-time PCR in control and c-myb siRNA-treated cells. The frequencies of (C) BFU-E and (D) CFU-E derived from CD34⁺ cells are presented as the mean from duplicate determinations from 3 independent experiments. White bars represent control siRNA-treated cells; black bars, c-myb siRNA-treated cells; dark gray bars, c-myb siRNA-treated cells cultured with exogenous NmU peptide; and light gray bars, c-myb siRNA-treated cells cultured with exogenous neurotensin.