Fig 2: Nectin-4 immunostaining of placental tissue sections. Polyclonal rabbit antibodies raised against the cytoplasmic domain of the human nectin-4 (a, b, d and e) or polyclonal goat antibodies raised against the C-terminal domain of the human nectin-4 (c and f) were used. Positive staining was apparent in the cytoplasm and cell membrane at the apical side of the syncytiotrophoblasts (arrows). Cytotrophoblasts were also positive (arrowheads, insets). The nectin-4 signals were much stronger in pre-eclampsia (d, e and f) compared with uncomplicated normotensive pregnancy (a, b, and c). Original magnifications a-f: 400 x. Scale bars, 100 µm

Fig 3: Nectin-4 immunostaining of tissue sections. Chorionic villi from early gestation was stained with polyclonal goat (a) or rabbit (b) antibodies against human nectin-4. Signals are observed in the syncytiotrophoblast (arrow) and cytotrophoblast cells (arrowhead). Maternal decidua was also examined using goat polyclonal antibodies against human nectin-4 (c and d). A subset of cells, possibly extravillous trophoblasts, is positive for nectin-4 staining (arrows). To detect cells of the trophoblast lineage, anti-pancytokeratin monoclonal antibodies were used (e and f). Original magnifications a-f: 400 x. Scale bars, 100 µm

Fig 4: Overexpression of the NCTIN4 gene in HTR-8 cell lines. a Invasion assay. Vertical axis indicates the ratio of cells invading through the matrigel membrane to those migrating through the control membrane. b NK cell cytotoxicity assay. Vertical axis indicates the percentage cytotoxicity calculated using an LDH cytotoxicity assay. Data obtained from control HTR-8 cells (open bars) versus HTR-8 cells overexpressing nectin-4 (black bars) were compared. Each bar represents the mean value. Error bars indicate the standard deviation

Fig 5: Semi-quantitative western blot analysis of nectin-4. a The same blot was incubated against a nectin-4 antibody (upper panel) and a ß-actin antibody (lower panel). Rabbit polyclonal anti-human nectin-4 antibodies we raised in this study were used. Lanes 1–4, lysates of placental tissue from uncomplicated normotensive pregnancies; lanes 5–7, lysates of placental tissue from pre-eclamptic pregnancies; lane R, recombinant nectin-4. The data of western blot using commercial goat polyclonal antibodies was shown in Additional file 1: Figure S1. b Semi-quantitative analysis of the blot described in A. Data obtained from uncomplicated normotensive pregnancy (open bars) versus severe pre-eclampsia (grey bars) were compared. The left and right panels indicate the data for the upper bands (open triangle), and lower bands (close triangle), respectively. Each bar represents the mean value. Error bars indicate the standard deviation