Figure 3.

Expression of ATP-binding pocket CLPB mutants impairs granulocytic differentiation. Cord blood CD34⁺ cells were transduced with lentivirus expressing the indicated CLPB cDNA (or empty vector control). GFP⁺ cells were sorted at 48 hours and then seeded into media containing G-CSF and SCF or methylcellulose containing G-CSF. (A) Lentiviral vector. (B) RNA expression of CLPB relative to β-actin messenger RNA. (C) CFU-G per 2000 GFP⁺ CD34⁺ cells. Percentage of mature neutrophils (D) and granulocytic precursors (E) on day 14 of culture; data are gated on CD14⁻ cells to exclude monocytes. (F) Percentage of caspase-3⁺ granulocytic precursors on day 7 of culture. (G) Cells were cultured for 7 days, and cell cycle was assessed by flow cytometry. Data represent 3-5 independent experiments. *P < .05, **P < .01, ***P < .005, repeated-measures 1-way ANOVA. IRES, internal ribosomal entry site; LTR, long terminal repeat; N.S., not significant; wPRE, woodchuck promoter responsive element.