Stat3F works as a dominant-negative mutant in TPO signaling.

(A) After 24-hour starvation, UT-7/TPO cells were transfected with luciferase reporter plasmid 4XAPREluc and pCAGGS vector or pCAGGS-Stat3F vector. After 12-hour culture, the cells were treated with TPO (hatched bar) or fetal calf serum alone (black bar) for 6 hours and then were harvested for luciferase assay. Values were normalized to transfection efficiency and represent the means of 3 independent experiments. (B) pCAGGS-Stat3F plasmids were introduced into UT-7/TPO cells, and 2 independent transfectants were established. Overexpression of Stat3F was confirmed by Western blot analysis using anti–HA-tag antibody. (C) Parental UT-7/TPO cells (○) and 2 lines of Stat3F expressing UT-TPO cells (clone 1, ●; clone 2, ▴) were cultured with 10 ng/mL TPO for the indicated periods. Cell numbers and viability were assessed by trypan blue dye exclusion.