Fig. 4.

S100A4 is needed for miR-325-3p mediated downregulation of osteoclast formation in CRC microenvironment. a OCPs were treated by DMEM or CT-26 CM for 4 days in the presence of RANKL and M-CSF. The protein level of S100A4 in OCPs were analyzed by using Western blot assay. b The relative intensity of S100A4 comparing with GAPDH in (a) was quantified (mean ± SD). c OCPs were transfected with agomir-325-3p or agomir-325-3p with plasmid containing S100A4 and cultured in CM for 4 days in the presence of RANKL and M-CSF. Relative TRAP activity in each group was analyzed (mean ± SE). d Representative images for osteoclastogenesis of OCPs cultured in CM after transfection with agomir-325-3p or agomir-325-3p with plasmid containing S100A4 in the presence of RANKL and M-CSF (Scar bar = 10 μm). TRAP positive multinucleated cells with more than three nuclei in each group were counted as mature osteoclasts. e The number of osteoclasts in (d) was qualified (mean ± SD). In vitro experiments were performed at least 3 times. *p < 0.05, **p < 0.01, ***p < 0.001