Ex vivo expansion of HSCs by cell-penetrating AML1a protein. (A) Schematic drawing of cell-penetrating AML1a protein (not to scale). (B) Immunoblotting of 32D cells treated with the cell-penetrating PTD-HA-AML1a protein with anti-HA antibody. The anti-tubulin blot allows comparison of the protein amount loaded. Asterisk (*) depicts degradation products. (C) Immunofluorescence staining of 32D cells treated with BSA (control) and PTD-HA-AML1a protein with anti-HA antibody. Nuclei are counterstained with propidium iodide (PI). Original magnification, ×200 (UPlan Apo 20×/0.70 objective). (D) DNA binding assays of nuclear extracts of cells treated with PTD-HA-AML1a protein. Nuclear extracts were incubated with biotinylated double-stranded oligo-DNA for the AML1-binding motif, or its mutant. The DNA-bound protein was visualized by immunoblotting with anti-HA antibody. WT indicates wild-type, and MT, mutont. (E) Schedule of PTD-HA-AML1a protein treatment. Bone marrow cells from Ly5.2 mice administered with 5-fluorouracil 4 days earlier were cultured with SCF, IL-3, and IL-6. The protein (10nM) was added 4 times a day (8°, 13°, 18°, 23°) for 4 days after prestimulation and transplanted into Ly5.1 recipient mice for CRU assay. (F) Results of 2 independent CRU assays of pretreatment (○) and after control (BSA; ▵) and PTD-HA-AML1a (●) treatments. Contribution of Ly5.2 donor cells to hematopoiesis in Ly5.1 recipient mice were analyzed 4 (experiment shown left) and 6 (experiment shown right) months after the transplantation.