1. Volume 21, Issue 5, Pages 1150-1159 (October 2017)
Fine-Tuned and Cell-Cycle-Restricted Expression of Fusogenic Protein Syncytin-2 Maintains Functional Placental Syncytia 
Xiaoyin Lu, Rui Wang, Cheng Zhu, Haibin Wang, Hai-Yan Lin, Yan Gu, James C. Cross, Hongmei Wang 
Cell Reports 
Volume 21, Issue 5, Pages (October 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 1150-1159DOI: (10.1016/j.celrep.2017.10.019)
Copyright © 2017 The Authors Terms and Conditions

3. Figure 1 Trophoblast Cell Fusion Is Accompanied by G0 Arrest
(A) Left: immunofluorescence of Ki67 in human placental villi at 7 weeks of gestation from the first trimester. CTB, cytotrophoblast cells; STB, syncytiotrophoblast. Scale bar represents 50 μm. Right: schematic representation of a cross-section through a human placental villus from the first-trimester placenta. Arrows indicate a layer of mononucleated CTB and a layer of multinucleated STB.
(B) Immunofluorescence staining of Ki67 and E-cadherin in BeWo cells treated with forskolin. Nuclei were stained with DAPI (blue). Scale bar represents 20 μm.
(C) Overview of the FucciNG system.
(D) A schematic overview of the experimental procedure. FSK, forskolin.
(E) Confocal images of cells treated as shown in (D). Scale bar represents 50 μm.
(F) Time-lapse imaging of cells treated as shown in (D), except that only mTagBFP2 was transfected. Still images from representative Movie S1 are shown; 0 hr corresponds to 03:59:59. Arrows indicate fusion (mTagBFP2-labeled blue cytoplasm flow). Scale bar represents 20 μm.
(G) Dye dilution assay for cell proliferation using CFSE. CFSE is a stable fluorescent cell staining dye that becomes diluted after mitosis. CFSE-bright cells indicate slow proliferation.
(H) Left panel: immunofluorescence with E-cadherin antibody. Nuclei were stained with DAPI (blue). Right panel: the fusion index of BeWo cells after forskolin treatment. Data are shown as mean ± SD of three experiments (∗∗p < 0.01). White dotted line circled area indicates the syncytia. Scale bar represents 20 μm.
See also Figure S1 and Movie S1.
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4. Figure 2
The Fusogenic Protein Syncytin-2 Is Highly Enriched in the G0 Phase, while Forced Expression of Syncytin-2 in Cycling BeWo Cells Enables the Fusion of S/G2/M Cells, which Leads to Unstable Syncytia with Mitotic-like Features
(A) Western blot of BeWo-FucciNG cells treated with/without forskolin (FSK). β-Actin was a loading control in all experiments.
(B) A schematic overview of the experimental procedure (left) and the western blot of the sorted cells (right). Note that Syncytin-2 is highly enriched in the red fraction.
(C) Localization of Syncytin-2 and Ki67 in human placental villi from 7 weeks of gestation by immunofluorescence. The area within the white dotted lines indicates the cytotrophoblast layer. The arrowhead indicates a cell that expressed Syncytin-2. Scale bar represents 50 μm.
(D) A schematic overview of the experimental procedure.
(E) Confocal images of cells treated as shown in (D). Scale bar represents 20 μm.
(F) Time-lapse imaging of cells treated as shown in (D), except that only mTagBFP2 and Syncytin-2 were transfected. Still images from representative Movie S2 are shown; 0 hr corresponds to 09:19:55. Arrows indicate where fusion occurred (mTagBFP2-labeled blue cytoplasm flow). Note that cells about to fuse contained green nuclei. Scale bar represents 50 μm.
(G) Time-lapse imaging of Syncytin-2-induced fused BeWo cells transfected with H2B-mCherry and mNG-farnesyl. Still images from representative Movie S4 are shown. 0 hr corresponds to 05:50:03. Arrows indicate a syncytium that underwent mitosis. Scale bar represents 20 μm.
See also Figure S2 and Movies S2, S3, and S4.
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5. Figure 3 Reduced Function of Syncytin-2-Induced Syncytia
(A) Syncytin-2-induced syncytia were less resistant to Listeria monocytogenes invasion compared to forskolin (FSK)-induced syncytia in BeWo cells. Scale bar represents 10 μm.
(B) Syncytin-2-induced cells produced less progesterone. Data are shown as means ± SD of three experiments (∗∗p < 0.01). Syn-2, Syncytin-2.
(C) Mitochondrial morphology. Fragmented mitochondria in FSK-induced, post-mitotic syncytia and elongated mitochondria in Syncytin-2-induced syncytia. White dotted line circled area represents syncytia. Scale bar represents 20 μm.
(D) Western blot analysis using indicated antibodies. The respiratory chain component Cox IV serves as a mitochondrial loading control.
(E) Localization of Syncytin-2 and Ki67 in human placental villi from a recurrent spontaneous abortion (RSA)-affected placenta and a gestation age-matched control by immunofluorescence. Arrowheads indicate cells that expressed Syncytin-2. Note the co-localization of Syncytin-2 and Ki67 in the RSA sample. Scale bar represents 50 μm.
See also Figure S3.
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6. Figure 4 Cell-Cycle Inhibitor p21 Regulates Syncytin-2 Expression
(A) Western blot of BeWo cells treated with or without forskolin (FSK).
(B) Western blot of control or p21 knockout BeWo cells. CTL, control cells that were transduced with lacZ-targeting sgRNA along with a CRISPR/Cas9 lentivirus. p21-KO, p21-knockout BeWo cells that were generated using the CRISPR/Cas9 lentivirus.
(C) Cell-cell fusion index was calculated for forskolin-treated indicated BeWo cells, using E-cadherin immunostaining. White dotted lines mark the syncytia (left panel). Red, E-cadherin; blue, DAPI. Scale bar represents 50 μm. Data are shown as means ± SD of 3 experiments (∗∗p < 0.01).
(D) p21-KO BeWo cells showed significantly lower expression of Syncytin-2 mRNA after forskolin treatment by real-time PCR. Data are shown as means ± SD of three experiments (∗∗p < 0.01).
(E) Luciferase assay of Syncytin-2 promoter activity. Data are shown as means ± SD of three experiments (∗∗p < 0.01).
(F) Upper panel: schematic illustration of the vectors that express the mNG-p21 fusion protein and luciferase reporter for Syncytin-2 5′-LTR activity. Lower panel: immunofluorescence to detect the expression of luciferase reporters. Blue: the nuclear-localized mTagBFP2 indicating the transfected reporter construct. Red: immunofluorescence using an anti-luciferase antibody. Scale bar represents 20 μm.
(G) Co-immunoprecipitation of p21 with GCM1.
(H) FluoPPI assay indicating the interaction between p21 and GCM1. Ash-p21, Ash (homo-oligomerized protein assembly helper)-tagged p21; ZG1-GCM1, ZG1 (homo tetramer ZsGreen1)-tagged GCM1. Scale bar represents 15 μm.
(I) Luciferase assay of Syncytin-2 promoter activity in transfected 293T cells. Data are shown as means ± SD of three experiments. a, b, c, d, different letters indicate statistical differences at p < 0.01, and the same letters indicate statistical differences at p > 0.05.
(J) Immunostaining showing localization of p21 and GCM1 or GCM1-NES in BeWo cells transfected with Flag-tagged p21 and HA-tagged GCM1 or GCM1-NES. NES, nuclear export sequence. Scale bar represents 15 μm.
(K) Association of p21 with Syncytin-2 promoter. BeWo-Flag-p21 (Tet-ON) cells were analyzed by ChIP assays in the absence of antibody (No Ab), presence of normal IgG, an anti-Flag antibody (Anti-Flag), or an anti-RNA polymerase II (Anti-RNA Pol II). The immunopurified DNA fragments were PCR amplified for promoter regions in the Syncytin-2 gene. As a control, a promoter region in the GAPDH gene was amplified.
(L) Localization of p21, GCM1, and Syncytin-2 in the human placental villi from 7 weeks of gestation by immunofluorescence. Arrows indicate cells that express both p21 and GCM1, or both p21 and Syncytin-2, respectively. Scale bar represents 50 or 20 μm (with the white boxes).
(M) Reconstructed z-stack confocal image representing higher resolution of the right panel of (L). Scale bar represents 20 μm.
See also Figure S4.
Cell Reports  , DOI: ( /j.celrep )
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