In vitro analysis of a + 23 Runx1 enhancer deletion and mutation series. (A) Six-species VISTA output for the + 23 mouse-frog conserved CNE. Conservation over more than 100 bp and more than 70% is indicated in pink. A deletion series was generated as shown (colored boxes; nucleotide positions of fragments in full-length enhancer are indicated). (B) Dual luciferase assays in 416B cells showed that all in vitro + 23 enhancer activity resides in the 235-bp central, most conserved segment (nt's 144-378). Luciferase activity (corrected for transfection efficiency) was normalized to the enhancerless pGL3P vector. Data are the mean (± SD) of at least 4 independent transfections, using at least 2 separately prepared batches of test plasmid. (C) The + 23 enhancer is dependent on intact Runx, Gata, and Ets motifs for its activity in 416B cells. Luciferase activity of mutated constructs (corrected for transfection efficiency) was normalized to the activity of the nonmutated + 23 enhancer. Data are the mean (± SD) of at least 6 independent transfections (apart from the constructs carrying a mutation in the Myb [n = 4] and single 3′Ets site [n = 3]), using at least 2 separately prepared batches of test plasmid. * and ** indicate significant changes from the nonmutated + 23 enhancer (2-tailed Student t test).