(A and B) Neutrophils derived from myeloproliferative neoplasm (MPN) patients were evaluated for expression of TGFβ family members by gene expression analysis (using Gapdh as reference gene). *P < 0.05 by unpaired Student’s t test. (A) Analysis of TGFβ1, TGFβ2, and TGFβ3 mRNA expression in an MPN cohort compared with healthy donors (Control). *P < 0.05 as compared with control group by unpaired Student’s t test. (B) TGFβ1 mRNA expression was determined in MPN patients stratified by disease subtype including essential thrombocytosis (ET, n = 47), polycythemia vera (PV, n = 28), and myelofibrosis (MF, n = 18). Data represent values relative to healthy control cells (Control). (C) qRT-PCR was performed for Tgfb1 mRNA using bone marrow mononuclear cells from MPL^WT and MPL^W515L mice (n = 6). ****P < 0.0001 by unpaired 2-sided Student’s t test with Welch’s correction. (D) Normal mouse bone marrow cells were directly transduced with retroviruses overexpressing either MPL^WT or MPL^W515L, following which cells were collected and Tgfb1 mRNA was measured by qRT-PCR. ****P < 0.0001 by unpaired 2-sided Student’s t test. (E–H) Representative contour flow cytometry plots of cells stained with antibodies against CD41 (clone HIP8) and CD42 (clone 1C2). GFP was simultaneously determined by flow cytometry and represents expression of GFP⁺ retroviral constructs (y axis, G and H). The total CD41⁺ cells (E and F) and GFP⁺CD41⁺ cells (G and H) in MPL^W515L spleens compared to MPL^WT controls. (I) Graph represents percentage of each population (CD41⁺, GFP^–, and GFP⁺) among live cells in the sample. Graphs (I and J) represent cells from spleens of 5 individual mice with the mean of the population indicated as a line (n = 5 MPL^WT and n = 5 MPL^W515L). Statistical significance determined using the Holm-Sidak method, with α = 5.000%. ****P < 0.0001. (J) Fold change in normalized Tgfβ1 mRNA expression of CD41⁺GFP⁺ cells vs. Tgfβ1 expression in CD41⁺GFP^– population from the same mouse (n = 3 MPL^WT and n = 3 MPL^W515L). *P < 0.05 by unpaired 2-sided Student’s t test. All error bars indicate the mean ± SD.