PMC

Osteoblast marker gene expression in SK11 cell cultures. (A–C) SK11 cells were subjected to differentiation conditions as described in Materials and Methods and the mRNA levels of indicated genes were measured between day 2 and day 9 by RT-qPCR (mean ± SD, n = 3). (D) SK11/Rx2^dox cells were treated with dox and subjected to RT-qPCR analysis of MGP mRNA.

Decreased RUNX2 stability as a function of osteoblast differentiation. Day-3 and Day-5 SK11/Rx2^dox cultures under osteogenic condition were treated with dox for 24 h and cell lysates were subjected to Western blot analyses with anti-FLAG antibodies between 0 and 12 h after dox withdrawal. Arrow indicates FLAG-RUNX2 in the top part. Bottom part shows Western blot analysis of Actin used as a loading control.

Loss of RUNX2 protein during osteoblast differentiation. (A–D) SK11 (A), ST2 (B), MC3T3-E1 (C), and NeMCO (D) cultures were subjected to Western analysis of RUNX2 on the indicated days. (E–G) SK11/Rx2^dox (E), ST2/Rx2^dox (F), and MC3T3-E1/Rx2^dox (G) cultures were treated with dox and subjected to Western analysis with anti-FLAG antibodies on the indicated days. Western analysis of Actin (SK11) and Coomassie Blue staining (other cell lines) were used as loading control. The arrow and the arrowhead indicate FLAG-RUNX2 and an endogenous RUNX2 isoform of approximately 55 kDa, respectively.

RUNX2 expression and mineralization in SK11 and SK11/Rx2^dox human embryonic progenitor-derived osteoblasts. (A) Day-3 SK11/Rx2^dox cell cultures were treated with 100 ng/ml dox for 24 h and subjected to Western blot analysis using either anti-FLAG (left panel) or anti-RUNX2 (right panel) antibodies. Arrow depicts the dox-inducible FLAG-RUNX2. Arrowhead in this and the following figures depicts an endogenous RUNX2 isoform of approximately 55 kDa. Additional RUNX2 isoforms detected by the anti-RUNX2 antibody are not labeled. Bottom panel shows β-Actin loading control. (B) SK11/Rx2^dox cells were treated with 100 ng/ml dox and subjected to ChIP assay with FLAG antibodies. Immunoprecipitated DNA was analyzed with primers () designed to amplify RUNX2 binding site at the osteocalcin promoter or an unrelated region as a negative control. (C) SK11/Rx2^dox cells were treated with 100 ng/ml dox and subjected to RT-qPCR analysis of BGLAP mRNA. (D) SK11/Rx2^dox or naïve SK11 cells were treated with 100 ng/ml dox and/or 10 ng/ml recombinant human BMP-2 from plating to the indicated day. Calcium deposition in representative wells is demonstrated by alizarin red staining.