Figure 5

MMP-13 potentiates OC differentiation. A. Western blotting analysis of MMP-13 expression in supernatants of wild type MDA-MB-231, scrambled shRNA and MMP-13 shRNA (clones #1, #2, #3 and #4) cells. B. PBMCs were primed with RANKL and M-CSF (primed pre-OC) for three days and then cultured with the addition of CM, obtained from wild type (MDA-MB-231 CM), scrambled shRNA (scrambled CM) and MMP-13 shRNA cells (clone #1) (MMP-13-shRNA CM) for further four days. The graph reports the mean ± SD of the number of multinucleated TRAP positive cells per field (20×). 10 fields for each experiment (n = 3) were examined. C. PBMCs were primed with M-CSF and RANKL for three days and then co-cultured with wild type, Scrambled and MMP-13 silenced MDA-MB-231 cells (clone #1) for further four days. Representative images of TRAP staining and their magnifications are reported. D. Recombinant human MMP-13 (10 ng/ml) was added alone or together with its specific inhibitor CL-82198 (10 μg/ml) to PBMCs primed with M-CSF and RANKL for three days. Representative images of TRAP staining of OC cultures at Day 7 are shown. The graph reports the mean ± SE of the number of multinucleated TRAP positive cells per field (20×). Five fields for each experiment (n = 3) were examined. *P < 0.05; NS, not significant. E. Western blotting analysis of galectin-3 in extracts of PBMCs cultured in the presence of RANKL and M-CSF for two, five or seven days (left panel) and of pre-OC cultured with CM as reported in (B) (right panel). F. Western blotting analysis for pro- and active MMP-9 forms in primed pre-OC treated as reported in (C) with silenced (sh#1 and sh#2) or Scrambled (scr#1 and scr#2) clones.