Fig. 2: Bone-secreted factors regulate SPP1 transcriptional activation.

A Experimental design for secretome analysis. After co-culturing PC3 and MC3T3, the conditioned media (CM) of each condition was centrifuged and analyzed by LC ESI/MS-MS. B Exclusion/inclusion criteria of differential proteins represented as a Venn diagram, where the numbers correspond to the proteins detected in each CM when LC ESI/MS-MS data were contrasted with murine protein database. Proteins in the CM the co-culture also found in the CM of PC3 cells alone were excluded in the following analyses. C Gene Ontology categories enriched considering the 65 murine proteins identified in the PC3/MC3T3 co-culture. The intensity of the color depicts the P value. The size of the dots represents the number of proteins identified for each biological process. D Experimental design of the cell adhesion assay. Commercial wells covered with a bone-like matrix (Corning, Cat. #3988) were treated with the CM from PC3 or MC3T3 cells growing alone, or from the PC3/MC3T3 co-culture. The preconditioning media were then removed and PC3 cells were cultured in the presence of the respective CMs, or with fresh culture medium. After 1 h, cells were washed with PBS and stained with crystal violet. Crystal violet was extracted with 10% acetic acid (V/V), and quantification of absorbance at 600 nm was performed Statistical significance was assessed via Kruskal–Wallis test. E Bar plot representing cell adhesion levels of PC3 cells to a bone-like matrix relative to control. F Protein-protein association analysis between OPN and the bone-secreted factors identified by LC ESI-MS/MS using STRING. OPN is represented as a red ball. OPN interactors are represented as gray balls with cherry borders. G Heatmap showing differential expression (GFOLD) of genes encoding OPN-related bone-secreted proteins assessed by RNA-seq in MC3T3 cells co-cultured with PC3 cells compared to MC3T3 cells cultured alone. The color scale goes from blue (GFOLD = −0.5) to cherry (GFOLD = 1). H SPP1 gene expression levels assessed by RT-qPCR in PC3 cells seeded in wells coated with type I collagen (i), fibronectin (ii) or coating mix (type I collagen and fibronectin; iii). PC3 cells seeded in untreated wells were considered controls (Ctrol). Values were normalized using PPIA as a reference gene and relativized to controls. Statistical significance was assessed via Wilcoxon test. Results are shown as mean ± S.E.M. Three independent experiments were performed. Statistical significance was set at P < 0.05. *P < 0.05, **P < 0.01.