Effects of TPO on primitive hematopoietic cell lines. (A) EML cells cultured with 100 ng/mL mSCF were treated with 100 ng/mL mTPO for the indicated time periods. Cell lysates were prepared, size fractionated, and probed for activation of p38 MAPK by Western blotting. (B) EML cells at a concentration of 1 × 10⁴ cells/mL were cultured with SCF alone (•) or with SCF plus 100 ng/mL hTPO (□). Viable cell numbers were monitored for the duration of the culture by trypan blue dye exclusion. Each point represents the average ± SD of triplicate samples; *P < .05. (C) After a 24-hour culture of EML cells with or without TPO, total RNA was prepared and subjected to real time RT-PCR for Hoxb4 quantification. The data represent the average ± SD of several independent experiments (n = 6, control; n = 4, TPO treatment, respectively). (D) UT-7/TPO cells were cultured with the indicated combination of cytokines for 24 hours, total RNA was prepared, and real-time RT-PCR was performed to quantify HOXB4 level. The bars represent the average ± SD of several independent experiments (n = 4, GM-CSF 10 ng/mL; n = 5, TPO 10 ng/mL; n = 7, TPO 100 ng/mL; n = 4, TPO 10 ng/mL plus GM-CSF 10 ng/mL).