The C-terminus of IGFBP2 is essential for IGFBP2's HSC supportive activity. (A) Schematic representation for IGFBP2 mutants. (B) WT, RGE267, and t248 IGFBP2 constructs were transfected into 293T cells and the levels of secreted IGFBP2 proteins in the media at 60 hours after transfection were evaluated by Western blot. (C) Normalized amounts of the WT and mutant IGFBP2 in the conditioned media (∼ 500 ng/mL) were added to STF medium, and then 20 CD45.1 donor Lin−Sca-1+Kit+Flk2−CD34− cells were cultured for 10 days. The cultured cells were cotransplanted with 1 × 105 CD45.2 total BM cells into CD45.2 recipients (n = 6). The data shown are representative of 2 independent experiments that gave similar results. (D) Total IGF-IR-null fetal liver cells (1 × 105) were transplanted into lethally irradiated WT or IGFBP2-null recipients. After 4 months, 1 × 106 total donor BM cells from primary WT or IGFBP2-null recipients were cotransplanted with 1 × 105 CD45.1 total BM cells into secondary CD45.1 recipients (n = 5). (E) Different donor lineages from long-term repopulation were determined. Representative data from 2 independent experiments that gave similar results are shown.
