Fig. 5: Differential processes of MPL activation.

A Time-dependent activation of MPL-downstream molecules in Ba/F3-HuMpl cells treated with ThC (1 μg/mL) and TPO (3 ng/mL). Phosphorylation of STAT5 and AKT was monitored at the indicated times after the addition of an agonist. Three independent experiments were performed, and similar results were obtained (see source data file). B Relative amount of MPL on the cell surface 0, 10, 30, 120, 240, and 360 min after the addition of ThC (1 μg/mL) and TPO (3 ng/mL) was measured with three independent experiments, bars indicate mean ± SD. Relative surface MPL at 240 and 360 min between ThC and TPO differed significantly, with P = 0.030 and 0.057, respectively, two-tailed paired t-test. For immunoblot data, see Supplementary Fig. 20. C Synergistic effects observed in each combination of agonists in the Ba/F3-HuMpl cell proliferation assay. Concentration–response curves for tested agonists in the absence or presence of fixed subactivation concentrations of ThC (0.1 μg/mL) (upper trace). The ratios of absorptions with and without ThC (0.1 μg/mL) are plotted (lower trace). EC₅₀ values for, with and without ThC are, 0.4 (95% CI of 0.2–0.7) and 2.8 (95% CI of 2.1–3.7) ng/mL, respectively. Non-liner fit variable slope with three parameters were used. D Proposed mechanisms of activation by two mechanistically discrete agonists, TPO and ThC: (I) Schematic depiction of MPL. Monomeric MPL has four N-glycosylation sites at N117, 178, 298, and 358; (II) ligand-bound monomeric state with each TPO and ThC; (III) ligand-bound nonactivated state; and (IV) dimeric signaling complexes.