YAP1 expression is regulated by GATA1 and contributes to MK maturation. (A) Fold changes in YAP1 mRNA (left) and protein expression (right) were assessed by real-time qPCR and western blotting on D10 and D12 compared with D7 during MK differentiation (n ≥ 3). (B) Fold changes in YAP1 mRNA (left) and protein expression (right) were assessed by real-time qPCR and western blotting after XMU-MP-1 (X, 300 nM) or peptide 17 (P, 2 μM) treatment (n ≥ 3). (C) Flow cytometry analysis of the percentages of CD41a⁺ MKs, CD42a⁺ MKs, and CD42a⁺ in CD41a⁺ MKs (n = 6). (D) Percentages of CD41a⁺ MKs (left) and CD41a⁺CD42a⁺ MKs (right) after YAP1 overexpression (PCDH-YAP1–labeled YAP1) or YAP1 knockdown (Plko.1-YAP1sh1/2–labeled YAP1sh1/2; n ≥ 6). (E) Fold changes in GATA1 mRNA levels in 13 patients with ITP relative to those in 14 healthy controls by real-time qPCR. (F-G) Assessment of GATA1/YAP1 mRNA expression in HEK293T cells after GATA1 overexpression (PCDH-GATA1–labeled GATA1) or knockdown (Plko.1-GATA1sh1/2–labeled GATA1sh1/2; n ≥ 5). (H-I) Representative immunoblots showing total GATA1 and YAP1 levels in HEK293T cells and the measurement of GATA1 and YAP1 levels based on GAPDH after GATA1 overexpression and knockdown (n = 4). (J) Measurement of luciferase activity to assess the binding activity of YAP1-WT (n ≥ 3). (K) Diagram depicting the domains of the YAP1 promoter with potential binding sequences (site 1, site 2, and site 3) for GATA1. Three mutants for the proposed binding sites (MT1, MT2, and MT3) were constructed. Binding site mutations (MT1, MT2, and MT3) are highlighted in red. (L) Measurement of luciferase activity to assess the binding activity of YAP1-MT1, YAP1-MT2, and YAP1-MT3 (n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. C, control; CDS, coding sequence; MT, mutant type; P, peptide 17; WT, wild type; X, XMU-MP-1.