Fig. 5

PCa-secreted GDF15 induces osteoclastogenesis. a BMMs from C57BL/6J mice were induced to differentiate toward osteoclasts with M-CSF and RANKL in the presence or absence of different concentrations of exogenous rhGDF15 (1 and 10 ng·mL⁻¹). After 7 days of culture, the cells were stained for TRAP and photographed by light microscopy to observe the multinucleated TRAP-positive cells (fused cells with 3+ nuclei) known as osteoclasts (left panel). Scale bars = 400 µm (upper) and 200 µm (lower). Quantification of multinucleated TRAP⁺ cells in different experimental groups (right panel). b To determine the effect of GDF15 on osteoclastogenesis, RT–qPCR analyses showing the mRNA expression of osteoclast markers (TRAP, cathepsin K, NFATc1, and carbonic anhydrase) using the same treatment regimen as in (Fig. 5a) were performed. BMMs from C57BL/6J mice were cultured for 7 days in the presence of RANKL and M-CSF and/or rhGDF15 along with c siRNA of CCL2 and CCL12 d and an anti-CCL2 antibody (10 µg·mL^-1), and the expression levels of TRAP transcripts were analyzed. e BMMs cultured in the presence of RANKL and M-CSF and/or rhGDF15 along with CM from C4-2B and LNCaP C-81 cells (parental and GDF15 KO cells). After fixation, the cells were stained for TRAP and photographed by a light microscope (left panel). Scale bar =200 µm, and the results are quantified in the right panel. The data represent the mean ± SEM. P ≤ 0.05 was considered to indicate statistical significance