IL-9 produced by osteoblasts promotes MK lineage cell expansion and platelet formation. (A) In situ hybridization analysis of IL-9 mRNA expression and hematoxylin and eosin staining of mouse tibia at 3 weeks. Scale bar = 100 μm. (B) IL-9 expression in the BM microenvironment. Mouse femur sections at 3 weeks were labeled with antibodies against OCN (red) and IL-9 (green). Blue, nuclear DAPI staining. Scale bar = 30 µm. (See supplemental Figure 7.) (C) IL-9 levels in BM of OSX-TSC1, OSX-Raptor, and control mice at 3 weeks (n = 6, Student unpaired t test). (D) Peripheral blood platelet count and MK numbers in OSX-TSC1 and control mice injected IP with recombinant murine IL-9 (1 µg/kg per day) for 14 days (n = 8, Student unpaired t test). (E) Quantification of FACS analysis of CD41⁺Sca-1⁺ cells proliferation (Sca-1⁺CD41⁺Edu⁺) in BM of IL-9–treated mice (n = 6, Student unpaired t test). (F) Peripheral blood platelet count and MK numbers in OSX-Raptor and control mice bilateral intratibially injected into the marrow cavity with purified IL-9 antibody (40 μg) for 14 days (n = 8, Student unpaired t test). (G) Quantification of FACS analysis of proliferation rate of CD41⁺Sca-1⁺ cell proliferation (CD41⁺Sca-1⁺Edu⁺) in BM of IL-9 antibody-treated mice (n = 6, Student unpaired t test). (H) Quantification of FACS analysis of proliferation rate of CD41⁺Sca-1⁺ cells (CD41⁺Sca-1⁺Edu⁺) cultured in conditioned medium from Raptor- or TSC1-deficient osteoblast cultures supplemented with IL-9 antibody or IL-9 (n = 3, Student unpaired t test). Data are representative of 3 independent experiments and are represented as mean ± SD. *P < .05; **P < .01; ***P < .001. OB, osteoblast.