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. 2011 Nov 22;21(11):1936–1947. doi: 10.1089/scd.2011.0422

FIG. 5.

FIG. 5.

DLX3 directly regulates expression of osteogenic markers in DFCs. Relative gene expression of ostegenic markers in DFCs was determined 48 h after transfection with (A) pDLX3 and pEV or (B) DLX3 siRNA(6) and NS siRNA by real-time PCR analysis. Sequences for primers and probes are listed in Table 1A. The gene expression of DFCs transfected with the respective controls (pEV or NS siRNA) was used for calibration. All values are means plus standard error (σ/√n) of 3 biological replicates. (C) Western blot analyses were done with RUNX2 and β-Actin specific antibodies and protein lysates of DFCs, 72 h after transfection with pDLX3 and pEV or DLX3 siRNA and NS siRNA in basal medium. (D) Chromatin immunoprecipitation analysis with DFCs (after DLX3 transfection) cultivated 72 h in basal medium, Dulbecco's modified Eagle's medium. The precipitations were done with either a DLX3 specific antibody or an unspecific antibody (immunoglobulin G, IgG) for control. PCRs for DLX3-binding sites on promoters of the genes RUNX2 and ZBTB16 were made with precipitated genomic DNA. ZBTB16, zinc finger and BTB domain containing 16; RUNX2, runt-related transcription factor 2; ALP, alkaline phosphatase; BSP, bone sialoprotein.