Cotransfection studies.

Mouse CCR2 promoter fragments were amplified by PCR of mouse genomic DNA using following primers: AAG ACA TGT ACC CTT TGT AGC (−269/−249), AAG ATG AAA GTT GAG AGG (−575/−558), TGT GTT AAA GTA CTT GCC GTG (−1428/−1408), and TTC CTT TGA TTC TGT GGT (+5/+22). We transfected human osteosarcoma U2OS cells with either cytomegalovirus promoter (CMV)-Foxm1b or control CMV-empty expression plasmids, as well as with luciferase (LUC) reporters driven by the mouse CCR2 promoter regions. CMV-Renilla was used as an internal control to normalize transfection efficiency. A dual luciferase assay (Promega) was performed 48 h after transfection, as described previously (22, 23, 29).