1. Volume 36, Issue 8, Pages 809-820 (August 2015)
Laminin α4 (LAMA4) expression promotes trophoblast cell invasion, migration, and angiogenesis, and is lowered in preeclamptic placentas 
N. Shan, X. Zhang, X. Xiao, H. Zhang, C. Tong, X. Luo, Y. Chen, X. Liu, N. Yin, Q. Deng, H. Qi 
Placenta 
Volume 36, Issue 8, Pages (August 2015)
DOI: /j.placenta
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2. Fig. 1
Dynamic LAMA4 Expression in Placentas. (A) Immunohistochemistry (IHC) of LAMA4 in human villi. (B) CK7 expression in human villi. (C and I) Negative controls with nonimmune rabbit IgG. (D) LAMA4 strongly expressed in human decidua in the first trimester. (E) HLA-G expression from the identical sample of human decidua. (F) CK7 expression in human decidua. (G) LAMA4 expression was moderate in the term placenta. (H) LAMA4 was rarely expressed in the preeclamptic placentas. Scale bar = 100 μm. Abbreviations: CTB, cytotrophoblasts; STB, syncytiotrophoblasts; EVT, extravillous trophoblasts, PE, preeclamptic.
Placenta  , DOI: ( /j.placenta )
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3. Fig. 2
LAMA4 Expression in Placentas and Maternal Serum. (A, B) LAMA4 expression was decreased in placentas of preeclamptics (N = 22) compared to healthy controls (N = 20) determined by Western blotting (n = 3, p < 0.01) and real-time RT-PCR (n = 3, p < 0.01). (C) LAMA4 expression in the maternal serum was not significantly different between preeclamptics (N = 20) and healthy controls (N = 20, p > 0.05). Abbreviations: PE, preeclamptic.
Placenta  , DOI: ( /j.placenta )
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4. Fig. 3
Expression of LAMA4, MMP2, MMP9, TIMP1, and TIMP2 after Various Treatments. (A) Expression of LAMA4, MMP2, MMP9, TIMP1, and TIMP2 were examined by Western blotting. (B, C) LAMA4 expression was decreased after various interventions as compared to the control group according to Western blotting and real-time RT-PCR. (D–G) Representative immunoblotting of MMP2, MMP9, TIMP1, and TIMP2 normalized to β-actin and comparisons of protein concentrations after various treatments.
Placenta  , DOI: ( /j.placenta )
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5. Fig. 4
Invasive and Migratory Abilities of HTR8/SVneo Cells Decreased with LAMA4 Silencing and H/R Conditions. (A, B) The invasive and migratory abilities of HTR8/SVneo cells decreased after exposure to H/R conditions and LAMA4 silencing (both n = 3, p < 0.001). (C, D) Statistical bar graphs of the invasion and migration assays. Data were analyzed by analysis of variance (ANOVA) to assess significant differences. Original images were taken at 200× magnification.
Placenta  , DOI: ( /j.placenta )
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6. Fig. 5
Effects of Different Treatments on HTR8/SVneo Cell Viability and Apoptosis. (A) Flow cytometric analysis of apoptosis across different treatments, including normal controls, H/R, LAMA4 silencing, and SB. (B) MTT assay showed no significant differences in viability after LAMA4 silencing and SB treatment by analysis of variance (ANOVA). (C) Significant differences in cell apoptosis were observed after H/R treatment (n = 3, p < 0.001).
Placenta  , DOI: ( /j.placenta )
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7. Fig. 6
Tube Formation of HTR8/SVneo Cells under Different Treatments. (A) Nontreated group. (B) siLAMA4 group. (C) H/R group. (D) SB group. Arrows indicate capillary-like structures. Bars = 100 μm. (E) Bar graphs showing quantification in the tube formation assay. The tube formation index was expressed as tube perimeter (mm) per area (mm [2]). **p < 0.01 compared with the nontreated control group.
Placenta  , DOI: ( /j.placenta )
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8. Fig. 7
Immunofluorescent Detection of LAMA4 Proteins and Nuclei in HTR8/SVneo Cells under Different Treatments. (A–C) LAMA4 expression (green) in normal control cells. (D–F) LAMA4 expression (green) decreased after siRNA transfection. (G–I) LAMA4 expression (green) decreased after 48 h of H/R. (J–L) LAMA4 expression (green) was not changed after SB treatment. Cell nuclei stained by propidium iodide (red) (n = 3 in triple). Original images were taken at 400× magnification.
Placenta  , DOI: ( /j.placenta )
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9. Fig. 8
Placental Villous Explants after 72-h Culture In Vitro. (A) Nontreated group. (B) siLAMA4 group. (C) H/R group. (D) SB group. Bars = 100 μm. (E) Graphical representation of trophoblast cell migration distance in placental villous explants (n = 5). **p < 0.01 compared with nontreated control group.
Placenta  , DOI: ( /j.placenta )
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10. Fig. 9
MAPK Signaling Pathway under Different Treatments. (A) Protein expressions of p-p38, p-JNK, and p-ERK were examined by Western blotting. (B–D) Representative immunoblotting of p-p38, p-JNK, and p-ERK normalized with β-actin and comparisons of protein concentrations after various treatments.
Placenta  , DOI: ( /j.placenta )
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11. Fig. 10
DNA Methylation of Placental LAMA4. CpG sites from +5312 to +5669 bps (relative to the transcription start site) of LAMA4 were included in the bisulfite genomic sequencing assay. A total of 33 CpG sites in the normal and preeclamptic (PE) placentas were included, respectively. Each square represents an individual CpG site with the yellow portions representing methylation-positive results and the blue portions representing methylation-negative results. The sizes of the yellow portions indicate fractional methylation. The data are presented as the mean values from the placental samples.
Placenta  , DOI: ( /j.placenta )
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12. Supplement 1
Invasive and Migratory Abilities of HTR8/SVneo Cells Decreased Under the H/R Conditions. The invasive, migratory, and tube formation abilities of HTR8/SVneo cells decreased after exposure to H/R conditions (n = 3 for both groups, p < 0.01). Bar graphs displaying data from the invasion, migration, and tube formation assays. Data were analyzed by independent t-tests to assess significant differences. Original images were taken at 200× magnification.
Placenta  , DOI: ( /j.placenta )
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