Fig. 4

PCa-secreted GDF15 augments osteogenic activity. a PCa factors promote osteoblast differentiation, as measured by alkaline phosphatase assay. MCOs were treated with different proportions of conditioned medium (CM 20%, 40%, and 80%) from the LNCaP C-81 and C4-2B cell lines (after 48 h of treatment) and subjected to alkaline phosphatase activity analysis. b Treatment with rhGDF15 for 48 h increased osteoblast differentiation, as determined by measuring alkaline phosphatase activity. c Representative alkaline phosphatase staining of MCOs after 10 days of treatment with the CM of LNCaP C-81 and C4-2B cells and different doses of rhGDF15. d, e Osteoblast differentiation (alkaline phosphatase activity) after 48 h of treatment with d CM obtained from PC3 and PC3 cells overexpressing GDF15 and e CM from C4-2B and LNCaP C-81 cells. f Assessment of mineralized nodules using alizarin red-S dye staining. MCOs were treated with the control, 20% CM from PCa cell lines, and/or rhGDF15 for 21 days, and the osteoblast differentiation medium was replaced every third day. The left panel shows representative alizarin red-S-stained nodules, and the right panel shows quantification of alizarin red-S staining with cetylpyridinium chloride (CPC) extraction. g MCOs were treated with the control or 20% CM from PCa cell lines and/or rhGDF15 for 72 h in osteoblast differentiation medium. Total RNA was harvested, RT–qPCR for osteogenic genes (alkaline phosphatase: ALP, Runx-2, Col-1a, and osteocalcin) was performed, and gene expression was normalized to that of β-actin. The data represent the mean ± SEM from three independent experiments. P ≤ 0.05 was considered to indicate statistical significance