Figure 3.

Influence of soluble DPP4 on activities of recombinant mouse CSFs in vitro, and effects of full-length and truncated CSFs alone and in combination on hematopoiesis in vivo in mice. (a) Influence of soluble DPP4 on activity of CSFs. Top, mouse bone marrow cells were treated with recombinant mouse GM-CSF, IL-3 or EPO which had been pretreated with PBS, soluble DPP4 or DPP4 first heat-treated at 56 °C for 1 h (DPP4-Δ*) to inactivate DPP4. Bottom, bone marrow was pretreated with diprotin A. Then, as indicated in the sequences shown on the y axis, cells were added to culture dishes with cytokines that had been first treated with PBS, DPP4 or DPP4-Δ* (as indicated in the first column in the sequence) and then with either PBS or diprotin A (second column); these treated cytokines were added with either PBS or the corresponding full-length cytokine (third column). *P < 0.05 compared to control medium (PBS, PBS, PBS) in that group. (b) Influence of full-length (FL) and truncated (T) recombinant mouse GM-CSF in vivo in WT and Dpp4^−/− mice on absolute numbers and cycling status of bone marrow HPCs. n = 4 mice per group. (c) Influence of full-length and truncated recombinant human erythropoietin in vivo on reticulocyte release to the blood of WT and Dpp4^−/− mice 18 h after a single (experiment 1, blood assessed 24 h later; n = 4 mice per group) or multiple injections of erythropoietin (experiments 2 and 3; n = 4 mice per group for each experiment). Experiment 3 used blood obtained from tail bleeds. The control treatment was PBS. (d) Influence of erythropoietin on absolute numbers of bone marrow HPCs in WT and Dpp4^−/− mice (n = 4 mice per group). The control treatment was PBS. For experiments 2 and 3 in c and for d, erythropoietin was given s.c. twice per day for 3 d at 10 U per injection, and mice were assessed 24 h after the last injection. For b–d, × = fold change from the indicated comparisons; *P < 0.05. All results are mean ± s.e.m.