The P106L mutation leads to ligand-independent growth and survival with maintained responsiveness to THPO, but defective THPO clearance. (A) Cell viability assay is shown using 0.4% Trypan blue solution. Depicted in panel A are (a) untransfected Ba/F3 cells and (b) Ba/F3 cells stably transfected with c-Mpl WT, (c) c-Mpl R102P, (d) c-Mpl F104S, (e) c-Mpl P106L, with and without the indicated treatment, respectively. Cells in log growth were plated at a density of 1.5 × 10⁶/well in 6-well plates in serum and IL-3-free Opti-MEM media and assayed for the number of viable cells on 5 subsequent days. Cells left untreated in Opti-MEM served as a negative control and cells stimulated with an additional 20 ng/mL IL-3 to the Opti-MEM media were used as a positive control for maximal growth. Directly after serum and IL-3 depletion, 50 ng/mL THPO was added to the indicated constructs. Results and standard deviations represent the median of 3 independent experiments. (B) THPO uptake analysis is shown from Ba/F3 cell culture supernatants. Cells in the log phase of growth were plated at a density of 2 × 10⁶ in 6-well plates in phenol-red-free RPMI 1640 media with 10% fetal calf serum. Four hours later, the cells were treated with THPO at a final concentration of 0.75 ng/mL. Thirty and 60 minutes after treatment, supernatants were harvested and assayed using a THPO enzyme-linked immunosorbent assay as described in “Materials and methods.” The results show the mean ± standard deviation of 3 independent experiments. (C) Immunoblot analysis is shown of the untransfected and stably transfected Ba/F3-cells 6 hours after IL-3 depletion and addition of serum-free Opti-MEM with 1% P/S. JAK2, RP-S6, ERK1/2, STAT5, STAT3, phospho-JAK2, phospho-RP-S6, phospho-ERK1/2, phospho-STAT5, and phospho-STAT3 were detected using the respective primary antibody as described in “Materials and methods.” Unstimulated cells after 6 hours IL-3 depletion in serum and IL-3-free media (lanes 1, 4, 7, 10, 13, indicated with an “×”) were compared with cells that were first IL-3 depleted for 6 hours and harvested after 30 (lanes 2, 5, 8, 11, and 14) and 120 minutes (lanes 3, 6, 9, 12, and 15) after THPO (50 ng/mL) stimulation, respectively. Equal loading was controlled with an antibody directed against tubulin for each protein preparation. Results are representative of 3 independent experiments. n.s., nonsignificant.