Figure 1.
Selected tissue sections of the FO region showing FO closure in the fetal and neonatal rat heart.
A. Closure and fusion of the FO during E20–P6. B. Immunohistochemical staining for nuclear cell proliferation marker PCNA (brown) at P2. Both PCNA and Control sections were counterstained with haematoxylin showing blue nuclei. Scale bars = 200 µm in A; scale bars = 50 µm in B. RA, right atrium; LA, left atrium; SP, septum primum; SS, septum secundum; FO, foramen ovale.
Figure 2.
Evidence of fibrosis during and after FO closure in rat heart.
A. H&E staining of FO region in neonates P4 and P7. Reduced cell density is shown by the arrows. Scale bars = 200. B. FAP mRNA expression during FO closure as determined by qRT-PCR. ***p<0.001, n = 3. C. Evidence of fibrosis in the remainder of FO region in adult rat hearts. a. H&E staining; b. Martius Scarlet Blue (MSB) staining. Scale bars = 500 µm. RA, right atrium; LA, left atrium; SP, septum primum; SS, septum secundum.
Figure 3.
Immunohistochemistry staining for Snail in rat heart tissue sections during FO closure.
Positive signals are brown colour by DAB staining. Scale bars = 50 µm. Negative control: sections were stained with secondary antibodies only.
Figure 4.
Immunohistochemistry staining for vimentin and PECAM1 in rat heart tissue sections during FO closure.
Double immunefluorescent staining was performed for vimentin (red) and PECAM1 (green). Scale bars = 50 µm. Nuclei were counterstained with DAPI (blue). Arrows indicate cells expressing both PECAM1 and vimentin. Negative control: sections were stained with secondary antibodies only.
Figure 5.
Immunohistochemistry staining of Notch pathway proteins during FO closure in rat heart.
Tissue sections of rat FO region were immunestained with Notch1, Notch3, Jagged1 and HRT1 using specific antibodies and visualised by DAB detection kit (brown). All sections were nuclear counterstained with haematoxylin (blue). Scale bars = 200 µm. SP, septum primum; SS, septum secundum. Arrows indicate highly expressed proteins.
Figure 6.
The mRNA expression of Notch1, Notch3, Jagged1 and HRT1 in the FO region of rat heart.
Total RNA of FO region was extracted from tissue samples obtained by laser microdissection and subjected to qRT-PCR. Results are presented as relative expression in comparison to E20. **p<0.01, ***p<0.001, n = 3.
Figure 7.
Activation of Notch3 in endothelial cells induces nuclear Snail expression. hCAECs were infected by adenovirus-N3ICD (A–C) or adenovirus vector-only (D–F).
48 hours after the infection the cells were double stained for Notch3 (red) and Snail (green). C and F, merged image with DAPI staining of the nuclei. Scale bar = 50 µm.
Figure 8.
Activation of Notch3 in endothelial cells induces cellular changes typical of EndMT.
A, Changes in cell morphology 7 days after infection (A-a), and the expression of vimentin and PECAM1 in N3ICD infected cells (A-b and A-c). Arrows in A-b indicate loss of membrane staining of PECAM1 in Notch3 positive cells; and arrows in A-f indicate membrane PECAM1. Arrows in A-c indicate cells exhibiting cell spreading. B, Western blotting for mesenchymal marker (vimentin) and endothelial markers (PECAM1) with β-Actin as a protein loading control. C & D, Quantitation of the protein expressions of PECAM1 (C) and vimentin (D). Data presented as the ratio to β-Actin, mean ± SE. *p<0.05, n = 3. Scale bar = 50 µm.