Progestins activate vascular endothelial growth factor gene transcription in endometrial adenocarcinoma cells  Michael D Mueller, M.D., Jean-Louis Vigne, Ph.D., Elizabeth A Pritts, M.D., Victor Chao, B.S., Ekkehard Dreher, M.D., Robert N Taylor, M.D., Ph.D.  Fertility and Sterility  Volume 79, Issue 2, Pages 386-392 (February 2003) DOI: 10.1016/S0015-0282(02)04577-6

FIGURE 1 (A), Ishikawa cells were transiently transfected with luciferase fusion vectors containing 2.3 kb of 5′-flanking DNA relative to the VEGF transcriptional start site with or without co-transfection of expression vectors encoding either human progesterone receptor A (hPRA) or B (hPRB). After recovery overnight, the cells were treated with ethanolic vehicle (control), R5020, R5020 and RU486, or phorbol-12,13-dibutyrate (12,13-PDB) for 24 hours. Error bars represent standard error of the mean (± SEM) from three different cultures. (B), Dose response of R5020 activation of the VEGF gene promoter in the presence of hPRB. Error bars show SEM among triplicates from a single representative experiment. Mueller. Progestins activate VEGF gene transcription. Fertil Steril 2003. Fertility and Sterility 2003 79, 386-392DOI: (10.1016/S0015-0282(02)04577-6)

FIGURE 2 Electrophoretic mobility-shift assay with oligonucleotides containing putative PREs (■, see Fig. 3 below) identified through the Transcription Element Search System (TESS) (13). Progesterone receptors expressed in reticulocyte lysates were added to the probes: PRE1: ACTAACTGTACAAAGACC, PRE2: GCTCTCTGTACATGAAGC, PRE 3: CTGGGCTGTTCTCGCTTC. Binding competition was performed by adding cold probe at a 100-fold excess. Mueller. Progestins activate VEGF gene transcription. Fertil Steril 2003. Fertility and Sterility 2003 79, 386-392DOI: (10.1016/S0015-0282(02)04577-6)

FIGURE 3 A series of constructs from the human VEGF gene promoter, including each of the PREs, (■), that were identified through mobility shift assay were created by digestion with restriction enzymes and cloned into the pGL2-promoter vector before transient transfection into Ishikawa cells in the presence of the hPRB expression vector. The cells were treated with ethanolic vehicle (control) or R5020. Error bars represent standard error of the mean (± SEM) from three different cultures. Mueller. Progestins activate VEGF gene transcription. Fertil Steril 2003. Fertility and Sterility 2003 79, 386-392DOI: (10.1016/S0015-0282(02)04577-6)