Fig. 2. RT-PCR analysis demonstrates that the PDL-L2 has gene expression identical to that of PDL cells in vivo. (A) MC3T3-E1 (U) represents MC3T3-E1 cells at 80% confluence; MC3T3-E1 (D) represents MC3T3-E1 cells at a fully differentiated stage (mineralization stage); PDL-L2 and NIH3T3 represent cells at confluence. Note that the expression level of periostin decreases with maturation of osteoblastic MC3T3-E1 cells, and it is higher in PDL-L2. The data are consistent with Fig. 1D. Runx2/Cbfa1/Osf2 is expressed in both PDL-L2 and MC3T3-E1 cells, whereas OCN and BSP are absent in PDL-L2 but present in MC3T3-E1. These are also consistent with Fig. 1E-G. (B) Nuclear extracts (N.E.) of PDL-L2 and MC3T3-E1 or cell lysates (C.L.) of C3H10T1/2 were electrophoresed on 8% SDS-PAGE and then subjected to immunoblot analysis with an antibody against Runx/Cbfa1/Osf2. Runx2/Osf2 is detected in the PDL-L2 cells, although its expression level is one-sixth of that in MC3T3-E1 cells. Since C3H10T1/2 cells do not express Runx2/Osf2, cells transfected with pcDNA3-Runx2/Osf2 were used for the control. Note that 20-fold dilution of cell lysate gave a comparable expression level of protein to the endogenous Runx2/Osf2 in the nuclear extract of PDL-L2. See text for details.