The dTMP-GH fusion protein strongly promotes thrombopoiesis. (A) Human cord blood-derived CD34⁺ cells were cultured with dTMP-GH (120 ng/ml) for 7, 10, and 13 days. (i) The proportion of CD41⁺CD42b⁺ cells was analyzed by flow cytometry. (ii) The expression levels of GATA-1, NF-E2, and β1-tubulin were analyzed by RT-PCR. (B) Histogram showing the proliferation of human cord blood-derived CD34⁺ cells cultured with dTMP-GH (120 ng/ml), dTMP (20 ng/ml), or rhGH (100 ng/ml) at day 7. The cells were counted by cytometer. Data are from 3 independent experiments. Error bars denote SD. *P < .05; **P < .01. (C) Histogram showing the number of proplatelet-forming megakaryocytes derived from human cord blood-derived CD34⁺ cells cultured with dTMP (20 ng/ml) or dTMP-GH (120 ng/ml) at days 8, 10, and 12. Data are from 3 independent experiments. Error bars denote SD. *P < .05; **P < .01. (D) Culture-derived platelets were stained with CD41-PE, CD42b-APC, and PI. Histogram shows the percentage of CD41⁺CD42b⁺PI⁻ culture-derived platelets detected by flow cytometry. Data are from 3 independent experiments. Error bars denote SD. **P < .01. (E) TEM analysis of blood- and culture-derived platelets (×30 000). (F) Aggregation of platelets. Platelets were stimulated with 2 U/ml thrombin for 15 minutes and analyzed by flow cytometry. Unstimulated platelets were used as control. Top and bottom panels represent blood-derived platelets and culture-derived platelets, respectively. (G) Platelets were stimulated with or without 2 U/ml thrombin for 15 minutes. Expression of P-selection was analyzed by CD62p-PE staining (blue line indicates unstimulated platelets; green line indicates thrombin-stimulated platelets). (H) Western blot analysis of p-STAT5 (i), and p-ERK1/2 (ii), in whole-cell lysates from M07e cells after exposure to dTMP (20 ng/ml) or dTMP-GH (120 ng/ml) for the indicated times. (I) Western blot analysis of p-Akt in whole-cell lysates from Meg-01 cells after exposure to dTMP (20 ng/ml) or dTMP-GH (120 ng/ml) for the indicated times.