FIG 2.

Regulation of hCGβ expression by GCM1. (A) GCM1 knockdown suppresses hCGβ expression in placental cells. BeWo cells stably expressing scramble or GCM1 shRNA were treated with dimethyl sulfoxide (DMSO) or 50 μM FSK for 24 h. Culture supernatants (medium) and cells (WCL [whole-cell lysate]) were harvested for immunoblotting with GCM1, hCGβ, and β-actin Abs. Note that secreted and glycosylated hCGβ has a higher molecular mass. In a separate experiment, cells were harvested for qRT-PCR analysis of hCGβ mRNA. (B) GCM1 knockdown suppresses hCGβ expression in placental explants. Human full-term placental explants were incubated with negative-control (neg. con.) or GCM1 siRNA, followed by immunoblotting with the indicated Abs or qRT-PCR analyses of GCM1 and hCGβ transcripts. (C) Complementation of hCGβ expression in GCM1 knockdown BeWo cells. Lentiviruses harboring an RNAi-resistant HA-GCM1 (HA-rGCM1) expression cassette were transduced into GCM1-knockdown BeWo cells treated with 50 μM FSK. Cells were harvested for immunoblotting with the indicated Abs. (D) Immunohistochemistry of hCGβ, GCM1, and TFAP2C in human placentas. First-trimester (8 weeks) and full-term placental sections were incubated with hCGβ, GCM1, and TFAP2C Abs, respectively. As a control, sections were incubated with normal rabbit serum (NRS) or normal mouse serum (NMS). Bar, 20 μm. Mean values and standard deviations obtained from three independent experiments are presented in panels A and B. *, P < 0.05; **, P < 0.01.