Runx2 regulates the secretion of soluble factors related to myeloma progression. (A) Cytokine array analysis of CM from NT control and Runx2 k/d 5TGM1 cells. Specific cytokines of interest (EGF, IL-9, OPN, SDF-1, VEGF) are identified by colored boxes. PC, cytokine array positive control. (B) The relative levels of cytokines in NT control (blue bars) and Runx2 k/d 5TGM1 cells (red bars) were normalized by comparison with the positive control signal. Levels of OPN, EGF, IL-9, SDF-1, and VEGF were significantly decreased in CM of Runx2 k/d cells. *Significant differences with corresponding P value. (C) OPN (left panel) ELISA of CM from Runx2 k/d and k/in 5TGM1 cells (red bars) compared with appropriate controls (blue bars) and RANKL (right panel) ELISA of CM from Runx2 k/in 5TGM1 cells (red bar) compared with control 5TGM1 cells (blue bar). Error bars represent mean ± SEM. Each sample was analyzed in duplicate. Significant differences between groups are indicated by P value. (D) Gelatin proteolytic analysis (zymogram) of MMP-9 activity in CM of Runx2 k/d MMIS and 5TGM1 cells compared with NT controls and Runx2 k/in 5TGM1 cells compared with vector control.