(A) Steady state levels of Runx-2 protein in MSCs from WT (lanes 1, 3, and 5) or Shn3^–/– mice (lanes 2, 4, and 6) were detected by Western blotting of MSCs cultured either with DMSO vehicle, Bzb, or lactacystin. In WT MSCs, p62 Runx-2 (red circle) was upregulated by Bzb treatment (lane 1 versus lane 3) and by lactacystin (lane 1 versus lane 5) to levels comparable to Runx-2 levels in Shn3^–/– MSCs (lane 2). Bzb and lactacystin also increased Runx-2 levels in Shn3^–/– MSCs, suggesting that Shn3-independent proteasomal activity targeting Runx-2 still remains functional in these MSCs. Bottom panel shows GAPDH used as a loading control. (B) C3HT1/2 cells were transfected with a luciferase construct containing 6 Runx-2–binding elements from the osteocalcin gene. Cotransfection of a Runx-2 expression construct (Rx-2) increased luciferase expression above baseline. Addition of either Bzb or lactacystin (third and fourth bars) further increased luciferase expression (*P = 0.0009; **P = 0.0003) compared with Runx-2–cotransfected cells without drug treatment. (C) MSCs from WT or Shn3^–/– animals were plated in osteogenic medium. Von Kossa staining (brown) revealed a dose-dependent increase in osteogenic activity with Bzb treatment in WT mice, while in Shn3^–/– mice, there was constitutively elevated von Kossa granule formation that was not increased by Bzb treatment. ^†P = 0.04; n = 4 wells. Original magnification, ×100. (D) In WT embryonic mesodermal fibroblasts (MEFs), treatment with Bzb showed an increase in expression of Alp and BSP, while in Runx-2^–/– cells, there was no response (upper 2 panels); however, expression of collagen I (bottom panel) was increased in both WT (^ΧP = 0.02) and Runx-2^–/– cells (^#P = 0.03). Stimulation of WT cells to promote Op formation with exogenous BMP-2 (two bars on far right of each panel) abolished the Bzb responsiveness of all 3 genes.