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. 2011 Jan 7;194(1):1–12. doi: 10.1159/000322035

Fig. 1.

Fig. 1

Smad4 is required for AVC endocardial cell EMT. a, b Representative brightfield photomicrographs of AVC explants incubated with control (a) or Smad4-targeted (b) siRNA. Scale bar = 20 μm. Cells scored as transformed have elongated, separated from adjacent cells, and entered the collagen pad (#). Many activated or transformed cells are evident in control explants and are greatly reduced in Smad4 siRNA incubated explants. Rounded, often contiguous cells are scored as epithelial cells (*). c Quantification of the number of cells in the collagen gel. Data are derived from 3 independent experiments normalized to control-high (HI) siRNA. Endocardial cells from AVC explants incubated either with no addition or with any of 3 independent scrambled control siRNAs with varying GC content [HI 62%, medium (MID) 38%, and low (LO) 29%] transformed on collagen gels. However, 2 independent siRNAs targeted against Smad4 inhibited endocardial cell EMT. Control-HI siRNA: normalized to 100%, control-MID siRNA: 97 ± 4.3% (mean ± SEM), control-LO siRNA: 103 ± 6.0%, no addition: 106 ± 7.7%, Smad4-A siRNA: 39 ± 2.8%, and Smad4-B siRNA: 36 ± 3.7%. Two-tailed Student's t test (control-HI vs. specific siRNA) control-MID: p = 0.549; control-LO: p = 0.706; no addition: p = 0.528; Smad4-A: p = 0.002, (* p < 0.05), and Smad4-B: p = 0.003, (* p < 0.05). The number of AVC explants examined and the number of cells in each category were as follows: control-HI (n = 24; total number of cells in gel 3,676), n = number of explants; control-MED (n = 24; total number of cells in gel 3,557); control-LO (n = 24; total number of cells in gel 3,763); no addition (n = 24; total number of cells in gel 3,880); Smad4-A (n = 24; total number of cells in gel 1,415), and Smad4-B (n = 25; total number of cells in gel 1,391). d, e Representative merged brightfield and fluorescent photomicrographs of ventricular explants. Scale bar = 20 μm. d Adenoviral introduction of GFP-only with the plane of focus at the surface of the collagen pad. Rounded, adjacent cells scored as epithelial cells (asterisks) are seen adjacent to the cardiac muscle. Elongated cells in the plane of the surface of the explant are termed activated (arrowhead). e Adenoviral introduction of TGFβR3 and GFP with the plane of focus in the collagen pad. Cells scored as transformed are elongated, separated from adjacent cells, and have entered the collagen pad (#). f Smad4 is required for TGFβR3-dependent, TGFβ2-stimulated ventricular endocardial cell EMT. The average percent of total GFP-expressing cells scored as epithelial, activated, or transformed is derived from 3 separate experiments. Two independent Smad4 siRNA constructs blocked TGFβR3-dependent EMT whereas control siRNA had no effect. g Smad4 is required for TGFβR3-dependent, BMP-2-stimulated ventricular endocardial cell EMT. The average percent of total GFP-expressing cells scored as epithelial, activated, or transformed is derived from 3 separate experiments. Two independent Smad4 siRNA constructs blocked TGFβR3-dependent EMT whereas control siRNA had no effect. For f and g, * denotes significance versus ligand-incubated GFP-only-expressing explants whereas # denotes significance versus ligand-incubated TGFβR3 and GFP-expressing explants. For the actual counts and statistical analysis of f and g, see online supplementary table 1.