Conditional inactivation of Tgfb1 in MKs in mice decreases TGF-β1 levels to ∼50% and increases MK number by ∼30% in BM. (A) BM from the femur and tibia was flushed, cells were lysed with lysis buffer, and TGF-β1 was measured by ELISA. (B) BM exudates were prepared by suspending BM cells in cold phosphate-buffered saline for 30 minutes on ice; levels of soluble TGF-β1 dissipated in the buffer were measured by ELISA (n = 3 from each group). (C) Representative images of H&E-stained bone section from PF4Cre;Tgfb1^flox/flox (PF4;Tgfb1) and WT (C57Bl/6) mice showing bone cells, including MKs indicated with green arrows. (D) The number of MKs in whole BM sections was counted manually (data supplement; supplemental Figure 1A). The MK number in PF4;Tgfb1 mice (n = 11) was ∼30% higher than that in controls (n = 10; P < .001), combining WT C57/Bl6 (n = 7) and littermate Tgfb1^flox/flox (n = 3) mice. (E) Representative images of bone section immunofluorescence stained with anti–platelet factor 4 (anti-PF4; green) and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) showing MKs (yellow arrows) in BM of PF4;Tgfb1 and WT (C57Bl/6) mice. (F) The number of PF4⁺ MKs per microscope field at 20× magnification (P = .02; n = 3).