Decreased expression of Wiskott–Aldrich syndrome protein family verprolin-homologous protein 2 may be involved in the development of pre-eclampsia  Juan Li, Xin Luo, Xiaoqiu Xiao, Xuemei Zhang, Hongbo Qi, Xiru Liu, Hua Zhang, Li Gao, Zhongmei Yang  Reproductive BioMedicine Online  Volume 28, Issue 1, Pages 70-79 (January 2014) DOI: 10.1016/j.rbmo.2013.07.015 Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Immunohistochemistry for WAVE2 protein concentrations and locations in normal (n=20) and pre-eclampsia placentas (n=20). (A) Representative immunostaining of WAVE2 protein in normotensive placentas (brown); WAVE2 protein located in the syncytiotrophoblasts (Sc, arrow) and vascular endothelial cells (Ec, arrowhead); (A inset), higher magnification view. (B) Representative immunostaining of WAVE2 protein in pre-eclampsia placentas; the positive signals for WAVE2 are lower in the pre-eclampsia trophoblast cells than in the normal trophoblast cells. (C) Negative control using non-immune rabbit IgG. Bars=25μm (A–C), 6.25μm (A inset). Reproductive BioMedicine Online 2014 28, 70-79DOI: (10.1016/j.rbmo.2013.07.015) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 WAVE2 expression in placentas and maternal serum in normal (n=20) and pre-eclampsia patients (n=20). (A) Ratio of WAVE2 mRNA concentration obtained from placentas (∗P<0.05). (B) Representative immunoblotting of WAVE2 normalized with GADPH (upper panel) and comparison of protein concentrations in different groups (lower panel; ∗P<0.05). (C) WAVE2 protein concentrations in maternal blood serum of patients with pre-eclampsia and healthy controls (∗P<0.05). Reproductive BioMedicine Online 2014 28, 70-79DOI: (10.1016/j.rbmo.2013.07.015) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 The correlation between WAVE2 and ROS concentrations in pre-eclamptic placentas. (A) Comparison of ROS concentrations in normal (n=20) and pre-eclampsia patients (n=20; ∗P<0.05. (B) Negative correlation between WAVE2 and ROS concentrations in pre-eclamptic placentas (r=−0.726, P<0.001). Reproductive BioMedicine Online 2014 28, 70-79DOI: (10.1016/j.rbmo.2013.07.015) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Effects of hypoxia–reoxygenation (H/R) on intracellular reactive oxygen species formation. (A) Representative picture showing a dramatic increase in fluorescence intensity in the H/R group compared with the control group. (B) ROS concentrations in the H/R and control groups (n=3; ∗P<0.05). DCFH=dichlorodihydrofluorescein diacetate. Reproductive BioMedicine Online 2014 28, 70-79DOI: (10.1016/j.rbmo.2013.07.015) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Effects of hypoxia–reoxygenation (H/R)-induced oxidative stress on proliferation, apoptosis and migration and invasion in HTR-8/SVneo cells. (A) The effect of H/R-induced oxidative stress on apoptosis in HTR-8/SVneo cells assessed by flow cytometry. (B) Mean percentage of early apoptosis from independent experiments; each experiment was run five times; significant differences were observed for H/R-induced oxidative-stress-mediated early apoptosis in HTR-8/SVneo cells. (C) Inhibition of proliferation in HTR-8/SVneo cells of the H/R group was significantly higher than that of the control group (the data in control group was 100%, n=3) (P<0.05). (D) Invaded cells in the invasion assay (upper panel) and summary of three independent experiments that assessed the effect of H/R-induced oxidative stress on invasion of HTR-8/SVneo cells (lower panel). (E) Migrated cells in the trans-well migration assay (upper panel) and summary of three independent experiments that assessed the effect of H/R-induced oxidative stress on migration of HTR-8/SVneo cells (lower panel). PI=propidium iodide. Bars=25μm. ∗P<0.05, ∗∗P<0.01. Reproductive BioMedicine Online 2014 28, 70-79DOI: (10.1016/j.rbmo.2013.07.015) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Expression of WAVE2 protein in HTR-8/SVneo cells exposed to hypoxia–reoxygenation (H/R). (A) Immunofluorescent detection of WAVE2 protein (green) and nuclei (red, propidium iodide, PI) in HTR-8/SVneo cells placed in H/R for 48h or unexposed. WAVE2 protein was located in the cytoplasm of HTR-8/SVneo cells, and cells demonstrated weaker staining after exposure to H/R for 48h compared with the control group (n=3). (B) Relatively reduced expression of WAVE2 protein in HTR-8/SVneo cells after exposure to H/R for 48h (n=3). (C) WAVE2 protein concentrations in the cell supernatant of the H/R and control groups, respectively (n=3). Bars=25μm. ∗P<0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Reproductive BioMedicine Online 2014 28, 70-79DOI: (10.1016/j.rbmo.2013.07.015) Copyright © 2013 Reproductive Healthcare Ltd. Terms and Conditions