Eltrombopag bypasses IFN-γ blockade of TPO intracellular signaling in human HSPCs. The phosphorylation state of STAT5, STAT3, AKT, and ERK was measured using phosphoflow at various times after treatment of human CD34⁺ cells with TPO (A-C) or eltrombopag (D-F) in the presence or absence of IFN-γ. (A) JAK-STAT5 pathway for TPO (n = 5). (B) AKT pathway for TPO (n = 5). (C) ERK pathway for TPO (n = 3). (D) JAK-STAT5 pathway for eltrombopag (n = 5). (E) JAK-STAT3 pathway for eltrombopag (n = 6). (F) ERK pathway for eltrombopag (n = 3). Insets are representative flow cytometry graphs from 1 donor for each signaling pathway. Results at each time point are displayed as percentages of maximum ± SEM in individual pathways. To underscore the impact of IFN-γ and the observed differences in magnitude of stimulation within each pathway between TPO and eltrombopag, percentages of maximum were calculated by dividing the MFI stimulated by TPO or eltrombopag, alone or in combination with IFN-γ, at each time point within a specific signaling pathway, by the maximum MFI measured within that pathway. Curve fit by polynomial nonlinear regression. *P < .05, 2-way ANOVA.