TGF-β1 and IL-4 worked synergistically to inhibit HPC colony formation. WT C57Bl/6 BM (5 × 104 cells per plate) was used for the colony assays. (A) HPC colony assays were performed where rTGF-β1 and rIL-4 were added to the plates at the indicated concentrations prior to incubation. When both cytokines were added, they were added in equal amounts. *P < .05, **P < .005, and ***P < .0005 when compared with media alone group as determined by Student t test. (B) CM obtained from pitpα−/−/β−/− MKs was pretreated with 1 µg/mL neutralizing TGF-β and/or IL-4 antibodies or their isotype controls for 1 hour at 4°C prior to being added to the HPC colony assays. Media alone, media with 10 ng/mL rTGF-β1, and media with 10 ng/mL rIL-4 were used as controls, each condition being treated with control or neutralizing antibody. *P < .05 and **P < .005 when compared with group with media vehicle pretreated with isotype control. ǂP = .019 when compared with group with pitpα−/−/β−/− MK CM pretreated with isotype control. (A-B) Total number of colonies was counted. Each group was plated in triplicate. Data are the mean colony numbers ± SEM. Ab, antibody.
