NIP-004 is a novel human c-Mpl activator. (A) Chemical structure of NIP-004. (B) Dose-dependent proliferation of UT-7/TPO cells, but not parental UT-7 cells, by NIP-004. Recombinant human TPO and rhIL-3 induced proliferation of UT-7/TPO and parental UT-7 cells, respectively. (C) TPO and NIP-004 induced proliferation of UT-7/EPO-HuMpl but not UT-7/EPO (top panel). EPO stimulated proliferation of UT-7/EPO and UT-7/EPO-HuMpl as a positive control. NIP-004 stimulated proliferation of Ba/F3-HuMpl but not Ba/F3-HuGCSFR (bottom panel). GCSF stimulated Ba/F3-HuGCSFR cells. TPO induced proliferation of Ba/F3-HuMpl cells as a positive control. IL-3 stimulated both Ba/F3-HuGCSFR and Ba/F3-HuMpl. Data in panels B-C are expressed as the mean ± SEM (n = 3 to 4) or mean ± SD (n = 2; Ba/F3-HuGCSFR) from independent duplicate experiments. (D) Induction of tyrosine phosphorylation of c-Mpl, Jak2, and STAT5 in UT-7/EPO-HuMpl cells by TPO and NIP-004. NC indicates negative control; E, 0.1 U/mL rhEPO; IL-3, 0.1 ng/mL rmIL-3; G, 10 ng/mL rhGCSF; T, 10 ng/mL rhTPO; NIP₁, 1 μg/mL NIP-004; NIP₂₀, 20 μg/mL NIP-004; P-Tyr, phosphotyrosine.