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. 2008 Jun 16;181(6):959–972. doi: 10.1083/jcb.200711044

Figure 5.

Figure 5.

Stable expression of CHIP inhibits osteoblast differentiation and mineralization. (A) Establishment of cell lines stably expressing HA-CHIP in MC3T3-E1 cells (#2 and #8). Endogenous CHIP and HA-CHIP expression was measured by immunoblotting with an anti-CHIP antiserum. (B) Stable expression of HA-CHIP in MC3T3-E1 cells reduces the endogenous Runx2 protein levels. The cell lines were treated with AA/β-GP for the indicated days. The mRNA (top) and protein (bottom) levels of Runx2 were measured by RT-PCR and Western blotting, respectively. (C) Stable depletion of CHIP using siRNA in MC3T3-E1 cells increased Runx2 protein levels. The cell lines with depletion of CHIP (#9 and #20) were treated with AA/β-GP for the indicated days. mRNA (left) and protein (right) levels of Runx2 were measured by RT-PCR and Western blotting, respectively. (D–F) CHIP inhibits the differentiation of MC3T3-E1 cells into osteoblast-like cells. CHIP- or CHIP siRNA–expressing cell lines were induced to differentiate into osteoblast-like cells in the presence of AA/β-GP for 4–21 d followed by the determination of AP activity (D), calcium accumulation by Alizarin red staining (E), and expression of the osteoblast marker genes (BSP and OCN) by real-time RT-PCR (F). Error bars represent SEM.