Fig 2.

Regulation of HtrA4 expression by GCM1. (A) GCM1 knockdown decreases HtrA4 expression. BeWo cells stably expressing scrambled or GCM1 shRNA were subjected to immunoblotting with GCM1, HtrA4, and β-actin Abs, respectively. In a separate experiment, cells were immunostained with HtrA4 Ab (green) and nuclei were stained by DAPI (blue), followed by confocal microscopy analysis. Note that HtrA4 signals in the cytoplasm were decreased in the GCM1 knockdown BeWo cells. (B) Expression of HtrA4, but not other HtrA family members, is decreased by GCM1 knockdown. BeWo cells stably expressing scrambled or GCM1 shRNA were harvested for quantitative PCR analysis of the transcript levels of HtrA family members. Means and SD obtained from three independent experiments are presented. (C) Overexpression of GCM1 stimulates HtrA4 expression. Mock and HA-GCM1-expressing JAR cells were harvested for immunoblotting and quantitative PCR analysis of HtrA4 protein and transcript levels, respectively. (D) Regulation of HtrA4 promoter activity by GCM1. Schematic representation of HtrA4 promoter region with the wild-type and mutant GCM1-binding site (GBS) is provided (top). 293T cells were transfected with pHtrA4-1kb or pHtrA4-1kbGBSmt with or without pHA-GCM1 expression plasmid (left). BeWo cells expressing scrambled or GCM1 shRNA were transfected with pHtrA4-1kb or pHtrA4-1kbGBSmt (right). At 48 h posttransfection, cells were harvested for luciferase assays. Means and SD obtained from three independent experiments are presented. (E) Interaction of GCM1 and the GBS in HtrA4 promoter. Recombinant GCM1-FLAG was incubated with radiolabeled HtrA4-GBS or HtrA4-GBSmt probes in the presence of GCM1 or syncytin-2 (Syn2) Ab in EMSA. The asterisk and arrow indicate the GCM1-FLAG-DNA complex and its supershifted complex, respectively. Association of GCM1 and HtrA4-GBS in BeWo cells was analyzed by ChIP using normal rabbit serum (NS) or GCM1 Ab for immunoprecipitation and designated primer pairs for PCR.