Effect of IL-7 and DKK1 on osteoblast formation and Runx2 activity. (A) Human BMMNCs were incubated in osteoblast differentiation medium as described in “Patients, materials, and methods” in the presence or absence of rhIL-7 (20 pg/mL-20 ng/mL) and DKK1 (20-100 ng/mL). CFU-F and CFU-OB formation was evaluated after 14 days with alkaline phosphatase staining and 21 days with alizarin red, respectively. Figures are representative of 3 independent experiments (original magnifications, × 1 [top] and × 5 [bottom]). Graphs represent the mean number of CFU-Fs and CFU-OBs/well ± SD of 3 independent experiments performed in triplicate. (^*P < .05; ^**P < .01). (B) Confluent human PreOBs were stimulated with or without IL-7 (20 pg/mL-20 ng/mL) or DKK1 (100 ng/mL) for 24 hours. After the culture period, nuclear extracts were obtained and Runx2/Cbfa1 binding activity and protein expression were evaluated by EMSA and Western blot, respectively, as described in “Patients, materials, and methods.” (C) Human BM cells were cocultured with XG-6 (on the top) in the presence or absence of blocking anti-IL-7 Ab (0.5 μg/mL) or anti-DKK1 Ab (5 μg/mL). The CFU-Fs were stained using alkaline phosphatase kit after 14 days as described in “Patients, materials, and methods.” Figures are representative of 3 independent experiments and graphs represent the mean number of CFU-Fs and CFU-OBs/well ± SD of 3 independent experiments performed in triplicate (^*P < .05). (D) BM cells were incubated with BM plasma (dilution = 1:2) of patients with MM (MM1, MM2, and MM3) in the presence or absence of a blocking anti-IL-7 Ab and CFU-F colonies were stained after 14 days. On the left is a representative experiment, with MM1 sample quantified by densitometry as total pixel intensity. Graphs on the right represent the mean number of CFU-Fs/well ± SD of an experiment performed in triplicate with 3 different MM samples (^*P < .05). Images were obtained as described for Figure 1.