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. Author manuscript; available in PMC: 2009 May 1.
Published in final edited form as: Biol Reprod. 2007 Dec 26;78(5):841–851. doi: 10.1095/biolreprod.107.065599

Fig. 6.

Fig. 6

GCM1 regulates PGF promoter activity and mRNA expression. A) Expression of exogenous GCM1 was confirmed by Western blot for HA-tagged GCM1. Cells were transfected with pHA-GCM1 or a control empty vector (pEF1/MycHis) and immunoblotted for anti-HA and β-actin. Paired lanes correspond to JEG-3, JAR, HeLa, and hEK-293, respectively. B) GCM1 increased transcriptional activity of the −1.5-kb clone in both trophoblasts and nontrophoblasts. Cells were cotransfected with pHA-GCM1 or pEF1/MycHis and (−1521/+34) PGF reporter construct. PGF reporter activity was determined 48 h later and fold activity above cells transfected with pEF1/MycHis for each cell type plotted. GCM1 overexpression significantly increased PlGF −1.5-kb promoter activity in both trophoblast and nontrophoblast cells (*, P < 0.05). C) GCM1 overexpression increased (*, P < 0.05) PGF mRNA expression in nontrophoblasts. GCM1 overexpression was accomplished with increasing concentrations of pHA-GCM1, and qRT-PCR was performed 48 h later to determine PGF and RPL32 mRNA expression. PGF mRNA expression was normalized to RPL32 expression, and relative changes in PGF mRNA expression were compared to control transfections with the pEF1/MycHis empty vector (set = 1). D) GCM1 overexpression restores PGF mRNA expression in hypoxic trophoblasts. JEG-3 cells were transfected with GCM1 overexpression plasmid (pHA-GCM1) or pEF1/MycHis and cultured under either 21% O2 or 1% O2 for 24 h. Quantitative RT-PCR was performed to determine PGF and RPL32 mRNA expression. PGF mRNA expression was normalized to RPL32 expression and relative changes in PGF mRNA expression were compared to the result of pEF1/MycHis transfected cells cultured at 21% O2 (set = 1). *, P < 0.05.