FIG. 3. .

A) Cyclic AMP stimulates GCM1 and ERVW-1 expression. Representative photomicrographs of Northern blot analysis of GCM1, ERVW-1, and RN18S mRNA levels following control (−) or 1 μM cAMP treatment (+) for 24, 48, and 72 h in wild-type Jeg3, αhCG/Cx43, JpUHD/Cx43, and JpUHD/trCx43 cell lines are shown. All probes detected a single band in each case: GCM1, 4 kb; ERVW-1, 3 kb; and RN18S rRNA, 2 kb. All cell lines showed a strong upregulation of GCM1 by 24 h of cAMP stimulation; ERVW-1 mRNA levels showed a slight increase at 24 h that was more strongly detected by 48 and 72 h of cAMP stimulation. B) GCM1 is upstream of Cx43. Real-time RT-PCR analysis of GJA1 mRNA levels in BeWo choriocarcinoma cells demonstrated that stimulation with 50 μM forskolin in the presence of nonsilencing RNA resulted in the upregulation of GJA1 mRNA levels. Silencing of GCM1 (inset) resulted in the inhibition of forskolin-stimulated GJA1 expression. Data are expressed relative to the level of GJA1 mRNA in the control samples and standardized against the geometric mean of three housekeeping genes, HPRT, YWHAZ, and RN18S rRNA. **P < 0.01, n = 4.