Figure 3

IL34 regulates BMP2-associated osteoblastic and osteoclastic differentiation. (A) Images of human mesenchymal stem cells differentiated into osteoblasts cultured in basic culture medium (CT-) or in osteogenic culture medium (CT+) in the absence or presence of BMP2 (10 ng/mL), IL34 (20 ng/mL) or combination of both at 10 and 21 days. Right panel: quantification of alizarin red staining. Magnification was similar for all views and the bar in CT- view at day 10 corresponds to 500 µm. (B) Real-time PCR quantification of early (RUNX2) and late (ALP and OCN) markers of osteoblastogenesis at days 0, 3 7 and 14. Data correspond to fold increase by 2^-ΔΔCt (cycle threshold) method. A representative experiment is shown. nd: non detected. (C) Western blot analysis of SMDA1/5 phosphorylation at different times of human mesenchymal stem cells differentiated into osteoblasts in basic culture medium (CT-) and in osteogenic culture medium (CT+) in the absence or presence of BMP2 (10 ng/mL), IL34 (20 ng/mL) or combination of both. (D) Differentiation of human CD14⁺ cells into osteoclastic cells analyzed by Tartrate Resistant Acid phosphatase activity (TRAP histoenzymology: purple staining) after 3-day culture period in the presence of MCSF (25 ng/mL) or IL34 (100 ng/mL), followed by an 8-day period of maturation with the addition of RANKL (100 ng/mL) and /or BMP2 addition (concentrations from 1 to 50 ng/mL) (E) Quantification of the different experiments repeated in triplicate and presented in D. At least two independent experiments have been carried in triplicate. *p<0.05, **p<0.01, ***p<0.001.