Gfi1, a transcriptional repressor, can repress the Runx2 promoter. (A) Various amounts of expression plasmid encoding wild-type mouse Gfi1 (WT mGfi1) were cotransfected (0-250 ng), balanced by empty pcDNA3.1 vector (EV; to a total of 250 ng) along with the −992/+111 mRunx2 promoter-pGL2 (250 ng) and pRL-TK Renilla (10 ng) into MC-4 cells using Lipofectamine. The relative luciferase units (RLU) of 3 independent transfections were determined, averaged, and plotted with the SD. We did not use the Renilla activity as a reference because it was sensitive to Gfi1. (B) Various amounts of TSA (as indicated) were added to MC-4 cells transfected with 250 ng p976 mRunx2 promoter-pGL2 and 250 ng of either WT mGfi1 or empty vector as in panel A. The ratios (Gfi1/EV) of the RLU expressed at each amount of added TSA were averaged from 3 independent transfections (each set containing independent duplicates) and the SD calculated. (C) Similarly, either WT mGfi1 or empty vector was cotransfected with a set of rat Runx2 5′-deletion series in pGL3 as indicated (3′ end of all is +1). Three independent transfections were averaged and the SD calculated. (D) The −108 to −1 mouse/rat Runx2-P1 promoter sequence is shown with putative Gfi1 cores underlined (the longer one contains 2 overlapped cores in opposite directions, thereby forming a palindrome) and asterisks above denoting the 3 base differences with the human. (E) Western blot of protein extracts from MC-4 cells stably transduced with puromycin-selectable retrovirus containing cDNA for (lane 1) empty, (lane 2) WT mGfi-1, and (lane 3) mGfi-1N382S were analyzed for Gfi-1 and Runx2 expression as well as β-actin. The ratio of Gfi-1 or Runx2 relative to β-actin calculated for each sample and compared with the ratio exhibited by cells transduced with the empty retrovirus and denoted below each blot.