Figure 2

Role of LCN2 in endothelial cell-osteoblast crosstalk.

(a) RT-PCR of Lcn2 in 1g- and 0.008g-treated mouse primary endothelial cells (upper panels) and mouse primary osteoblasts (lower panels), normalized versus Gapdh. (b) Western blot analysis of LCN2 protein in 1g- and 0.008g-treated mouse primary endothelial cells (upper panels) and mouse primary osteoblasts (lower panels) treated with 1g- and 0.008g-EC-CM, normalized versus actin. (c) RT-PCR of Lcn2 in mouse primary osteoblasts subjected to 1g and 0.008g in the presence of 1g- and 0.008g-EC-CM, normalized versus Gapdh. Wild-type (WT) and LCN2 KO osteoblasts were treated with 1g- or 0.008g-EC-CM. (d) MTT proliferation assay. (e) Representative images of ALP cytochemical staining. (f) Densitometric quantification of ALP staining. (g) RT-PCR of Rankl in 1g- and 0.008g-EC-CM treated mouse primary osteoblasts, isolated from calvarias of WT and LCN2 KO mouse, normalized versus Gapdh. Total bone marrow cells were isolated from the long bones of WT and LCN2 KO mice and cultured with 1g- or 0.008g-EC-CM. (h) Representative images of TRAcP staining. (i) Quantification of TRAcP positive multinucleated cells per field. (j) RT-PCR of the LCN2 upstream activators IL-1β, Tnfα and IL-17 in 1g- or 0.008g-treated mouse primary endothelial cells, normalized versus Gapdh. Bone marrow cells (BM) were analyzed as technical positive control for Tnfα and IL-17. (k) ELISA assay for IL-1β in EC-CM from mouse primary endothelial cells exposed for 96 hours to 1g, 0.08g or 0.008g. Images are representative and data are the mean ± SD of at least 3 independent experiments (Student’s t test). All gels have been run under the same experimental conditions. Actin was evaluated on the same blotting of LCN2. Bar = 100 μm.