FIGURE 3.

Astakine 1 stimulates proliferation and differentiation into the semigranular cell lineage of Hpt cells both in vitro and in vivo. A, Hpt cell cultures were supplemented with r-astakine 1 or r-astakine 2 every second day. After 1 week, RT-PCR was conducted to monitor the expression of PCNA (proliferation marker of Hpt cells), KPI (gene marker for semigranular cells), and SOD (gene marker for granular cells). The addition of astakine 1 could stimulate transcription of PCNA and KPI significantly. The effect of r-astakine 1 and r-astakine 2 is detectable from ∼3 nm (4.5 ng/well) and is optimal at a concentration of ∼65 nm (∼100 ng/well), whereas a toxic effect to the cells is obtained at ≥120 nm. B, r-astakine 1 mainly stimulates the synthesis of semigranular cells in vivo. Relative hemocyte counts for total hemocytes, semigranular cells, or granular cells were calculated as hemocyte number 24 h postinjection/initial hemocyte number. r-astakine 1 injection induced a significant increase in the number of total hemocytes, mainly due to an increase of semigranular cells. The columns represent the mean of three separate experiments; the error bars indicate S.D. values.