1. Volume 46, Pages 92-101 (October 2016)
DNA methylation-associated repression of MEST/PEG1 expression contributes to the invasion of extravillous trophoblast cells 
Wei Peng, Ying Chen, Xin Luo, Nan Shan, Xi Lan, David Olson, Hua Zhang, Yu-Bin Ding, Hong-Bo Qi 
Placenta 
Volume 46, Pages (October 2016)
DOI: /j.placenta
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2. Fig. 1
Immunohistochemical detection of MEST expression in different types of trophoblast cells in human first-trimester villi. Extravillous trophoblasts (EVTs) were strongly stained and cytotrophoblasts (CTBs) were weak detected by the MEST antibody (A) as indicated by the HLA-G antibody (B). The CTBs and syncytiotrophoblasts (STBs) were indicated by cytokeratin 7 (CK7) antibody staining (C). D. Negative control (NEG) with nonimmune rabbit IgG. E. Quantification analyzed of relative levels of MEST in EVTs, CTBs and STBs. EVTs: extravillous trophoblasts; STBs: syncytiotrophoblasts; CTBs: cytotrophoblasts. IOD: integrated optical density. Scale bar = 200 μm.
Placenta  , DOI: ( /j.placenta )
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3. Fig. 2
Differential expression of MEST in the EVT cell lines HTR-8/SVneo and HPT-8 and the CTB cell line JEG-3. A. Western blotting detecting MEST expression in different trophoblast cell lines, results are presented (mean ± SEM, n = 3) in the bar graph, p<0.001 compared with JEG3 group. β-actin was used as internal control. B. Immunofluorescence detection of MEST in HTR8/SVneo cell line. C. Immunofluorescence detection of MEST in JEG-3 cell line. Fluorescence signals specific to MEST antibody are visualized as green, and the nuclei are shown by DAPI staining (blue). Bar = 200 μm.
Placenta  , DOI: ( /j.placenta )
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4. Fig. 3
The effects of knocking down MEST on EVT migration and invasion. A. Western blotting detection of MEST/PEG1 knock down by small interference RNA (siRNA) specific to MEST gene (si-MEST) for 48 h. si-Ctrl, control siRNA or scramble siRNA; (n = 3 in triplicate, t-test, **p < 0.01 compared with the si-Ctrl group). B. Matrigel assay detecting the influence of knocking down MEST on invasion and migration in HTR8/SVneo cells. Scale bar = 200 μm. (n = 3 in triplicate, t-test, ***p < 0.001 compared with the si-Ctrl group). C. Real-time detection of migration and proliferation using xCELLigence assay in HTR8/SVneo cells following induction of si-MEST or si-Ctrl. FBS+ and FBS- indicate the lower chambers filled with medium with or without 10% fetal bovine serum (FBS), respectively.
Placenta  , DOI: ( /j.placenta )
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5. Fig. 4
The effects of knocking down of MEST on villous explants outgrowth and migration. A. The outgrowth and migration (indicated by the black arrows) of villous explants in si-MEST group and si-Ctrl group after 72 h culture, the white dotted line indicate the migratory front and villous tips; B. Graphical representation of trophoblast cells migration distance in placental villous explants. (n = 3 in triplicate, t-test, **p < 0.01 compared with the si-Ctrl group).
Placenta  , DOI: ( /j.placenta )
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6. Fig. 5
The effects of knocking down of MEST on the expression of N-cad, Vimentin and their regulator Twist were examined by Western blotting, **p < 0.01 compared with the si-Ctrl group. (n = 3 in triplicate, t-test).
Placenta  , DOI: ( /j.placenta )
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7. Fig. 6
Protein expression differences between missed abortion and normal controls. A. Western blotting detecting the expression of MEST, N-cad, Vimentin, Twist in the first trimester villi of missed abortion and normal controls; B. Vertical scatter plot indicating the corresponding protein expression in first trimester villi from missed abortion and normal controls, p < 0.05 means significant differents between the two groups. (n = 7 in triplicate, t-test).
Placenta  , DOI: ( /j.placenta )
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8. Fig. 7
The different levels of MEST/PEG1 promoter DNA methylation in the first trimester villi from missed abortion and normal controls. A. The gene structure of MEST/PEG1 isoform 2 was obtained from the UCSC Genome Browser. B. Promoter sequence of the MEST gene used in bisulfate sequencing PCR (BSP). C. Representative bisulfite-sequenced promoter region of MEST isoform 2. Unmethylated cytosines in CpGs are converted to uracils and detected as thymines, whereas methylated cytosines in the CpGs remain unchanged. D. Individual results for methylation status at each CpG site. The percentage of hypermethylated CpG was calculated using a 2-sided Fisher's exact test. Each row represents a single DNA clone. Each circle represents a single CpG site. Filled and open circles indicate methylated and unmethylated cytosines, respectively. CpG, cytosine-phosphate-guanine.
Placenta  , DOI: ( /j.placenta )
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9. Fig. 8
Correlation between MEST/PEG1 methylation levels and protein expression in missed abortion villi and controls. (Pearson's correlation, p < 0.01).
Placenta  , DOI: ( /j.placenta )
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