Figure 6.

Inhibition of MetAP2 affected human HSPCs proliferation, differentiation, and engraftment potential. (A) Relative expression of MetAP2 in CB cells revealed by Q-RT-PCR between CD34⁺ fraction and unfractionated CB MNC (of which ∼ 99% cells are CD34⁻). MetAP2 expression in unfractionated CB MNC was normalized to 1. N = 8. (B) GAPDH pI shifting assay showing Met-excised GAPDH protein (red arrowhead) with a greater pI and Met-unexcised (black arrowhead) GAPDH in control or fumagillin-treated CB CD34⁺ cells with a lower pI. (C) Fold change of cell number after 3 days of DMSO (control) or fumagillin treatment (at 10nM or 1μM) of CB CD34⁺ cells. A total of 0.1-0.25 million cells were treated with vehicle 10nM or fumagillin for 3 days. After treatment, viable cells were enumerated by trypan blue exclusion. N = 5 and the cell numbers at day 0 were normalized to 1. (D) Total number of colonies formed in colony-forming assay between control and fumagillin treated CB CD34⁺ cells. After 3 days of treatment, 3000 cells (from vehicle) and cell numbers proportional to its expansion (fumagillin-treated) were cultured in methylcellulose medium H4434 without fumagillin for 14 days and total colonies numbers were counted. N = 3. (E) Lineages of colonies in colony-forming assay. Images of colonies acquired 14 days after cells plated (top). Erythroid colonies and granulocyte/macrophage colonies were enumerated and their ratio of were shown (bottom). N = 3. (F) BM engraftment of CB CD34⁺ cells after vehicle or fumagillin treatment in NOD/SCID mice at 6 weeks after transplantation. Human cells engraftment was determined by human CD45-positive, mouse CD45.1-negative fraction. The comparisons were evaluated by the Student t test. (G) Western blot comparing the level of β-catenin, total and phospho-ERK1/2 between control and fumagillin treated CB CD34⁺ cells normalized with β-actin. (H) Relative CamKII activity in CD34⁺ CB cells treated with vehicle, 10nM or 1μM fumagillin for 3 days.