Neutralizing TGF-β with antibodies in vitro and in vivo reversed the myelosuppressive effect of pitpα^−/−/β^−/−MKs. (A) HPC colony assays were performed in the presence of media with 10 ng/mL rTGF-β1 or CM from pitp WT and pitpα^−/−/β^−/− MKs that was pretreated with 1 µg/mL anti-TGF-β neutralizing antibody or an isotype antibody control for one hour at 4°C prior to being placed into the HPC colony assays. BM from WT C57Bl/6 mice was used for the HPC colony assays. The number of CFU-GM (Ai), BFU-E (Aii) and CFU-GEMM (Aiii) was counted per 5 × 10⁴ nucleated BM cells. Each group was plated in triplicate. Data are presented as mean ± SEM and are representative of 2 separate experiments. ***P < .0005 when compared with media isotype control group as determined by Student t tests. (B-C) Pitpα^−/−/β^−/− and WT mice were treated with intraperitoneal injections once per day for 2 days of either 0.5 mg/kg anti-TGF-β or isotype control antibodies. (B) Twenty-four hours following the final injection of antibodies, BM was collected and analyzed phenotypically for LT-HSC (Bi), ST-HSC (Bii), and MEP (Biii) numbers per femur by flow cytometry. (C) BM was also collected and analyzed by HPC colony assay. The number of CFU-GMs (Ci), BFU-Es (Cii), and CFU-GEMMs (Ciii) per femur was counted (n = 3 per group). For the colony assays, each mouse sample was plated in triplicate. For the in vivo data, data represent mean ± SEM. *P < .05 and **P < .005 when compared with pitpα/β WT isotype control as determined by Student t test.