Figure 4.

Expression of SUPYN and cell fusion indices in primary, spontaneously syncytializing human cytotrophoblast cells derived from TS21 and normal gestational age-matched control placentas. (a) ERVH48-1 transcription in primary trophoblast cells was quantitated using RT-PCR. Fold-induction ratio of ERVH48-1 transcripts in TS21 were compared with gestation matched control placenta at 4 h. (b) Detection of SUPYN protein in primary trophoblast cell culture supernatants by a SUPYN-specific ELISA (ng/ml). Experiments were performed in duplicate using 3 independent placental samples. Statistical analysis was performed using the Mann Whitney U test. Statistical significance was set at * : p < 0.05, ** : p < 0.01. Values represent mean ± SD (n = 3). (c) Time course of SUPYN protein expression in cultured primary, syncytializing cytotrophoblast cells from TS21 and gestational age-matched control placentas. Immunofluorescence images were obtained at 4 h, 48 h and 72 h after plating [SUPYN (red), E-cadherin (green) and Hoechst 33342 (blue)]. Upper panels are of representative cultured primary cells from gestational age-matched control placentas; lower panels are of representative cultured primary cells from TS21 placentas. Scales are as indicated. (d) The number of nuclei in SUPYN-expressing cells was counted and fusion indices were calculated. Experiments were performed using three independent samples with 5 independent microscopic fields evaluated for each sample. Values represent mean ± SD (n = 3). Statistical analysis was performed using the Mann Whitney U test. Statistical significance was set at * p < 0.05. (e) Expression of CGB transcripts from TS21 and gestational age-matched control placentas. Quantitative analysis of CGB gene expression in TS21 cells was compared to gestational age-matched control placental cells at each time point. Statistical analysis was performed using the Mann Whitney U test. Statistical significance was set at * p < 0.05.