Fig. 4. 92/546 acts as a strong DPP response element in COS cells. The expression of luciferase under the control of the lab550 enhancer or derivatives thereof was analyzed in COS cells after co-transfection with combinations of MAD-, MEDEA- and TKVQD-expressing plasmids. Cells were transfected with 4 µg of the reporter plasmids lab1-550luc (A) or its derivatives, schematically represented at the bottom of the graph, and (B) co-transfected with 2 µg of each indicated expression plasmids. enhancerless: parental luciferase plasmid pt81-luc. The amount of transfected DNA was kept constant (10 µg) by addition of psG5 plasmid. Bars represent the luciferase activity of transfected cell extracts (mean±s.e.m. of three to ten independent experiments, each carried out in duplicate), expressed as -fold activation over the basal activity of the reporter construct. Values were normalized by co-transfection of 0.1 µg of a pCMV-ß-gal plasmid as an internal standard. lab550 activity is increased 70-fold after the co-transfection of all four plasmids. (A) Although MAD and MEDEA together also stimulated expression of lab550 (14-fold), the addition of TKVQD further increased expression fivefold; TKV did not stimulate lab550 expression in the absence of co-transfected MAD and MEDEA. In contrast to lab550, the activity of HOMRE (B) was increased only threefold by DPP signaling. DPPRE activity was increased 84-fold and the activity of DPPRE carrying mutation 300, which allowed for a strong DPP response in the endoderm, was increased 32-fold. Thus, mutation 300 did not show a stimulatory effect in COS cells but rather somewhat reduced the DPP response. The reduced induction of 92/546m300 when compared with 92/546 was due to a higher basal level of 92/546; the measured activity levels after induction were similar (data not shown).
