Figure 2.

Secretome from mechanically‐stimulated MLO‐Y4 cells reduces the differentiation of human monocytes into osteoclasts by a mechanism dependent on the primary cilium and PTH1R. MLO‐Y4 cells were serum‐deprived for 24 h, treated with 1 mM aqueous chloral hydrate or with 100 nM PTHrP (7‐34) for 1 h and stimulated with shear stress (10 dynes/cm²) or with 100 nM PTHrP (1‐37) for 10 min. CM was collected after 18 h. To evaluate the differentiation of monocytic cells into osteoclasts, human monocytes were treated with 20 ng/ml M‐CSF and 20 ng/ml RANKL plus the corresponding 20% MLO‐Y4 cell‐conditioned mediums. Then, cells were fixed, permeabilized, and stained with hematoxylin. The differentiation of human monocytes into osteoclasts was determined by evaluation of the morphology, observing the formation of giant cells (≥50µm) with at least three or more nuclei. Representative images of each condition are shown (a). The percentage of cells with three or more nuclei evaluated with ImageJ software is represented (b). Results are the mean ± SD of triplicates. *p < 0.05 versus SC; **p < 0.01 versus SC; ^a p < 0.05 versus corresponding PTH1R inhibition; ^b p < 0.01 versus corresponding cilium or PTH1R inhibition. CM, conditioned media; FF; fluid flow; M‐CSF, macrophage colony‐stimulating factor; PTHrP, PTH‐related protein; PTH1R, PTH 1 receptor; RANKL, receptor activator for nuclear factor κ B ligand; RANKL and (+) α‐MEM medium; SC, static control; SD, standard deviation.