Figure 2.

Methotrexate reduces splenomegaly. (A) Weight of individual spleens from 10–11 week old mice of the indicated genotypes shown relative to wild-type controls treated with phosphate-buffered saline carrier control (PBS, gray), methotrexate (MTX, green) or ruxolitinib (rux, orange) for 28 days. Samples were compared by one-way ANOVA. (B) Representative spleens from mice of the indicated genotypes and treatments immediately after dissection showing differences in size and reduction in spleen size following MTX treatment. Scale bar is 5mm. (C) Hematoxylin and eosin stained sections through formalin-fixed spleens from mice of the indicated genotypes treated with the indicated compounds for 28 days. Images were obtained from a Zeiss Axioskop 2 with 5×/0.15NA (top row, scale bar is 1mm) and 20×/0.5NA (lower row, scale bar is 100μm) objectives, a MicroPublisher 5.0 RTV camera and Qimaging v3.1.3.5 software. Brightness and contrast were adjusted in Photoshop CS5. (D–G) Predicted interactions of methotrexate with JAK2 occludes the ATP-binding site. (D) Illustrated representation of the human JAK2 JH1 (kinase) domain (from 5TQ8.pdb). Methotrexate (magenta sticks) is predicted to bind between the N and C lobes of the kinase in the ATP-binding site (cyan sticks). (E) Molecular surface of kinase with ligands bound (carbon gray, nitrogen blue and oxygen atoms shown in red). (F) MTX showing predicted H-bonding (orange dashes) to labelled residues within the binding site and ion-pair interaction with lysine 882. Residues highlighted in F and G are within 5 Å of the bound ligand. (G) View of bound MTX as in (F) rotated 90° about the Y axis.