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. 2019 Dec 20;9:19502. doi: 10.1038/s41598-019-55933-x

Figure 3.

Figure 3

Cell fusion and expression of placental fusion-related proteins and hormones in primary spontaneously syncytializing peri-term human cytotrophoblast cells with and without forced SUPYN expression. (A) Expression of fusion-associated genes in primary trophoblast cells using semi-quantitive RT-PCR. Gels have been cropped for clarity, conciseness and comparison. Full length gel images for Fig. 3A are included as Supplementary Fig. S9 in the Supplementary Materials and Methods. (B) Detection of SUPYN protein in primary trophoblast cell cultures by a SUPYN-specific ELISA. Data depict relative fold change compared with 3H timepoint (cell-associated values normalized to 3H cell-associated baseline and cell supernatants to 3H cell supernatant). Upper panel, cell associated SUPYN; Lower panel, SUPYN in supernatants. Experiments were performed in duplicate using 4 independent placenta samples. Values represent means ± SDs (n = 4). (C) Time course of SUPYN protein expression in cultured primary peri-term cytotrophoblast cells. The upper panels show phase contrast images at each time point after plating, the lower panels an overlay fluorescence immunocytochemical analysis of an adjacent field [SUPYN protein (red), E-cadherin (green) and nucleus (blue)]. Scale bar = 50 micrometers. (D) Cellular morphologic changes after transient FLAG-tagged SUPYN (SUPYN) and control, FLAG-tagged ZAG (ZAG) overexpression in cultured primary peri-term cytotrophoblast cells. Anti-FLAG antibody (red) detects SUPYN-expressing cells and anti-ZO-1 (green) detects cell boundaries. Nuclei are stained blue. Expression of SUPYN and ZAG were detected by an anti-FLAG antibody, and the number of nuclei in SUPYN- or ZAG-expressing cells were plotted. (E) The number of nuclei in SUPYN- or ZAG-expressing cells was counted and fusion indices were calculated. Experiments were performed three independent times. Values represent means ± SDs (n = 3). Paired t-tests were used for statistical comparisons. Statistical significance (*p < 0.05). SUPYN–suppressyn; ASCT2–Alanine, Serine, Cysteine Transporter 2, the syncytin 1 receptor; MFSD2–major facilitator superfamily domain-containing protein 2, the syncytin 2 receptor; hCG–human chorionic gonadotropin; ZO-1–zona occludens-1.