Figure 1

Establishment of C4-2B/Rx2^dox sub-line with conditional Runx2 expression. A, Schematic diagram of the pSLIK-based lentiviral vector. Initially developed for expression of shRNAs [39] the pSLIK vector was used in the present study to express Runx2. The Hygromycin resistance marker (Hyg) and the Dox-dependent activator protein rtTA3 are constitutively expressed under the control of the Ubi-c promoter. Upon treatment with Dox, rtTA binds to its tetracycline responsive elements (TRE) and drives expression of the inserted cDNA (Flag-Runx2). The Runx2 block diagram depicts its glutamine/alanine-rich QA domain, the DNA-binding Runt domain, and the proline/serine/threonine-rich PST domain. Arrowhead indicates the position of the R265D and R268D mutations in Runx2-M, which eliminate Runx2's DNA binding function. B, Whole cell extracts, prepared from C4-2B/Rx2^dox and C4-2B/Rx2-M^dox cells treated with the indicated concentrations of Dox, were subjected to western blot analysis using anti-Flag antibodies. C, Total RNA was extracted from C4-2B/Rx2^dox cells treated with Dox or vehicle, as well as from PC3^high cells, and the mRNA levels of Runx2 (and GAPDH as control) were measured by RT-qPCR. D, Whole cell extracts were prepared from C4-2B/Rx2^dox cells treated with Dox as indicated and from ROS 17.8/2 osteoblastic cells [41], and subjected to western blot analysis using anti-Runx2 antibodies. The same blot was re-probed with anti-Tubulin antibodies as loading control. E, C4-2B/Rx2^dox and C4-2B/Rx2-M^dox cells were transiently transfected with the 6XOSE2-luciferase reporter plasmid and subjected to luciferase assay. Dotted line represents the background luciferase activity with no cell extract. F, C4-2B/Rx2^dox cells were treated with Dox and levels of the indicated transcripts were measured by RT-qPCR and corrected for that of GAPDH. In Figure 1, bars represent Mean ± SEM (n = 3) from a representative experiment, which was repeated at least three times with similar results. Abbreviations used: Dox, Doxycycline; Veh, vehicle; BSP, Bone Sialoprotein; MMP9, Matrix Metalloprotein-9; OC, Osteocalcin.