1. Volume 35, Issue 3, Pages 163-170 (March 2014)
Extravillous trophoblast cell invasion is promoted by the CD44–hyaluronic acid interaction 
H. Takahashi, T. Takizawa, S. Matsubara, A. Ohkuchi, T. Kuwata, R. Usui, H. Matsumoto, Y. Sato, H. Fujiwara, A. Okamoto, M. Suzuki, T. Takizawa 
Placenta 
Volume 35, Issue 3, Pages (March 2014)
DOI: /j.placenta
Copyright © 2013 Elsevier Ltd Terms and Conditions

2. Fig. 1
In situ expression of CD44 and HA in the human first-trimester placenta. (A–C) Double immunostaining for CD44 (green in A) and HLA-G (red in B) in the decidua. A 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; blue)-stained, merged, image of the same section is shown in C. EVT cells that are HLA-G-positive express CD44 (arrows). (D–F) Double immunostaining for CD44 (green in D) and cytokeratin 7 (KRT7; red in E) in a floating villus. A DAPI (blue)-stained, merged, image of the same section is shown in F. Villous cytotrophoblast (arrowheads) and syncytiotrophoblast (arrows) are CD44-negative. (G–I) Double immunostaining for HA (red in G) and CD31 (green in H) in the decidua. A DAPI (blue)-stained, merged, image of the same section is shown in I. Note the remarkable gradient of HA staining toward the perispiral arterial regions; the spiral arterial endothelial cells are CD31-positive (arrows). Bars = 100 μm.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions

3. Fig. 2
CD44 expression by EVT cells and EVT cell lines. (A) Western blotting for CD44 in isolated EVT cells and first-trimester CV. CD44s, the most common form of CD44; CD44v, variant isoforms. ACTB was used as an internal control. (B) Western blotting for CD44 in EVT (HTR8/SVneo and HChEpC1b) and VT (BeWo and JEG-3) cell lines. ACTB was used as an internal control. (C) Real-time PCR analysis of CD44 mRNA levels in EVT cells in collagen (type 1)- and Matrigel-coated dishes. EVT cells were replated for 4 h in collagen-coated dishes and then transferred into collagen- or Matrigel-coated dishes. After culture for 12 h, CD44 expression levels in the cells were examined. The expression level of Matrigel was set to 1.0. CD44 expression levels were normalized to that of GAPDH. (D) Real-time PCR analysis of CD44 mRNA levels in EVT cell lines in collagen- and Matrigel-coated dishes. The expression level of Matrigel was set to 1.0. CD44 expression levels were normalized to that of GAPDH. Data are means ± SD of the results from at least three independent experiments. (E) Western blotting for CD44 in EVT cell lines in collagen- and Matrigel-coated dishes. GAPDH was used as an internal control.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions

4. Fig. 3
Epigenetic regulation of CD44 in EVT cells and EVT cell lines. (A) Locations of the 34 CpG sites (sites 1–34) in CD44 promoter and exon 1 regions. (B) The rate of demethylation change at each CpG site in EVT cells compared to CV. Methylation status was analyzed using bisulfite genome sequencing. Individual DNA clones (34–62 in number) of genomic DNA from isolated EVT cells and CV from first-trimester placental tissues (n = 5; 8–10 weeks of gestation) were sequenced. The rate of demethylation change was calculated using the following formula; [(extent of unmethylation in EVT cells/extent of unmethylation in chorionic villi)–1] × 100. Date is present as the mean value from the five placental samples. (C) The methylation status of each of the 34 CpG sites in EVT cells (white bars) and CV (black bars). The extent of demethylation at each CpG site was calculated using the formula: [extent of unmethylation/(extent of unmethylation + extent of methylation)] × 100. Date is present as the mean value from the five placental samples. (D) The methylation status of VT and EVT cell lines. Individual DNA clones (n = 13–21) of genomic DNA of EVT and VT cell lines were sequenced. The extent of demethylation at each CpG site of each cell line is shown.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions

5. Fig. 4
HA-dependent invasion by EVT cells and EVT cell lines. (A) Transwell invasion by EVT cell lines (HTR8/SVneo and HChEpC1b) and a VT cell line (JEG-3) in the presence of 5, 50, and 250 μg/ml HA in the lower chamber. Data are presented as numbers of invading cells relative to such numbers in the absence of HA. The extent of cell invasion in the absence of HA was set to 1.0. HA regulates cell invasion in a dose-dependent manner. (B) Transwell invasion by isolated EVT cells in the absence or presence of HA (250 μg/ml). Data are means ± SD of the results from at least three independent experiments. Student's t-test or Tukey's test; *p < 0.05.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions

6. Fig. 5
HA/CD44-dependent invasion by EVT cells and EVT cell lines. (A) Western blotting evaluation of CD44 protein levels in EVT cell lines transfected with siRNAs. Two siRNA molecules targeting CD44 (siCD44-#1 and siCD44-#2) and negative control siRNA (Ctrl) were used. GAPDH served as an internal control. The intensity of each band was densitometrically quantified using LAS-4000 and Multi Gauge 3.1 software (Fujifilm). The intensities of CD44 bands were normalized to those of GAPDH. (B) Transwell invasion by EVT cell lines transfected with siCD44s. Data are presented as numbers of invading siCD44-transfected cells relative to the numbers of invading cells transfected with Ctrl in the presence or absence of HA (250 μg/ml). The invasion level of Ctrl in the presence of HA was set to 1.0. (C) Real-time PCR analysis of CD44 mRNA levels in invading siCD44-transfected EVT cells in the presence of HA (250 μg/ml). The expression level of Ctrl was set to 1.0. CD44 expression levels were normalized to that of GAPDH. (D) Transwell invasion by EVT cells transfected with siCD44s. Data are presented as numbers of invading siCD44-transfected cells relative to those of cells transfected with Ctrl in the presence of HA (250 μg/ml). The invasion level of Ctrl was set to 1.0. Data are means ± SD of the results from at least three independent experiments. Tukey's test; *p < 0.05, **p < 0.01.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions

7. Fig. 6
MMP9 expression in EVT cells and EVT cell lines treated with siCD44s. (A) Real-time PCR analysis of MMP9 mRNA levels in invading siCD44-transfected cells in the presence or absence of HA (250 μg/ml). The expression level of Ctrl in the presence of HA was set to 1.0. MMP9 expression levels were normalized to that of GAPDH. (B) Real-time PCR analysis of MMP9 mRNA levels in EVT cells and first-trimester CV. The expression level of CV was set to 1.0. MMP9 expression levels were normalized to that of GAPDH. Data are means ± SD of the results from at least three independent experiments. Student's t-test or Tukey's test; *p < 0.05, **p < 0.01.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions

8. Supplemental Fig. 1A
Real-time PCR analysis of CD44 mRNA levels in siRNA-transfected EVT cell lines (HTR8/SVneo and HChEpC1b). Two siRNA molecules targetting CD44 (siCD44-#1 and siCD44-#2) and negative control siRNA (Ctrl) were employed. EVT cell lines were exposed to different concentrations of siCD44s (1, 5, 10, 30, and 60 nM). CD44 mRNA expression was significantly inhibited in the concentration range of 1–60 nM in HTR8/SVneo; CD44 expression was significantly downregulated in the concentration range of 10–60 nM in HChEpC1b. The expression level of Ctrl was set to 1.0. CD44 expression levels were normalized to that of GAPDH. Data are means ± SD of the results from three independent experiments. Dunnett's test; **p < 0.01. (B). Cell proliferation of HTR8/SVneo and HChEpC1b cells. Cells (5000 cells/1 00 μL) were plated in 96-well plates and then transfected with siCD44 at 30 nM. After 24 h, cell growth was assayed using CellTiter-Glo (Promega, USA) according to the manufacturer's protocol. The luminescence was read with GlomaxMulti (Promega). The level of siRNA Ctrl was set to 1.0. Data are means ± SD of the results from three independent experiments. There were no significances in the two cell lines. Dunnett's test.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions

9. Supplemental Fig. 2
Morphometric analysis of hyaluronic acid (HA) in the decidua shown in Fig. 1G and I. Representative fluorescence intensity from hyaluronic acid (HA) in the portion of the spiral artery indicated with an arrow in G. The fluorescence intensity of HA was analyzed using the linescan function of MetaMorph image analysis software. Briefly, a line was drawn from an endothelial cell (EC) to the stroma away from the artery in G. The average intensity of HA (red) was determined for each point along the line and graphed as intensity vs pixel distance along the line. (G) Immunostaining for HA (red). (I) Double immunostaining of HA (red) and CD31 (green). A DAPI (blue)-stained, merged, image of the same section is shown in I. Bars = 100 μm.
Placenta  , DOI: ( /j.placenta )
Copyright © 2013 Elsevier Ltd Terms and Conditions