Fig. 5

Bone-metastatic prostate cancer cells promote osteoclastogenesis by secretion of S100A4. a Pre-osteoclasts (pOCs) received PC3- or mtPC3-CM and were then tested for osteoclast generation. Representative tartrate-resistant acid phosphatase (TRAP)-stained images (left panels) and quantification of TRAP+ multinucleated cells (MNCs) with 3 or more nuclei (right panel). Scale bar = 100 μm. ***p < 0.001 by unpaired two-tailed Student’s t-test. b pOCs were treated with 30% PC3-CM or mtPC3-CM with BSA or OPG (100 ng/mL). Representative TRAP-stained images (left panel) and quantification of TRAP+ MNCs (right panel). Scale bar = 100 μm. ***p < 0.001 by one-way ANOVA with post hoc Tukey’s test. c pOCs receiving the indicated percentages of CM from mtPC3-C or mtPC3-S cells were TRAP-stained and then counted for TRAP+ MNCs. ***p < 0.001 by one-way ANOVA with post hoc Tukey’s test. d pOCs receiving 30% mtPC3-C-CM or mtPC3-S-CM were cultured on dentin slices. Representative confocal images of dentin surfaces (left panels) and values of pit depth and resorbed area (right panels) are presented. Scale bar = 100 μm. ***p < 0.001 by unpaired two-tailed Student’s t-test. e Western blots of c-Fos, NFATc1, and β-actin in BMM and pOCs after the indicated number of days with 30% mtPC3-C-CM or mtPC3-S-CM. f pOCs were tested with 30% mtPC3-CM, together with the indicated concentrations of control antibody or 4A. TRAP+ MNCs were counted. ***p < 0.001 by one-way ANOVA with post hoc Tukey’s test. Data were presented as the mean ± SD. The full-length gels are presented in Supplementary Fig. 3 g