The Par6c pathway is required for BMP-2-stimulated, TGFβR3-dependent ventricular endocardial cell EMT. a–d ALK5 is required for AVC and TGFβR3-dependent ventricular endocardial cell EMT. a ALK5 inhibitor blocks BMP-2-stimulated, TGFβR3-dependent EMT. Average percent of total GFP-expressing cells scored as epithelial, activated or transformed. Means are derived from 3 separate experiments. All ventricular explants were given 5 nM BMP-2. GFP served as a negative control to determine the basal level of transformation. TGFβR3 induced statistically significant increases in transformed cells with a concomitant decrease in epithelial cells. This effect was abolished in the presence of ALK5 kinase inhibitor SB431542. b ALK5-targeted siRNA inhibits AVC endocardial cell EMT. Quantification of AVC endocardial cells migrated into collagen gel. Data are derived from 3 independent experiments normalized to control (LO) siRNA. Endocardial cells from AVC explants given control siRNA transform on collagen gels, whereas 2 independent siRNA constructs targeted to ALK5 inhibit transformation. Control siRNA: normalized to 100%, ALK5-A siRNA: 31 ± 1.2%, and ALK5-B siRNA: 27 ± 0.5%. Two-tailed Student's t test (control vs. treatment) ALK5-A: p = 0.0003 (*** p < 0.001) and ALK5-B: p = 0.000042 (*** p < 0.001). The number of AVC explants examined and cells in each category were as follows: control (n = 30; total number of cells in gel 4,694), n = number of explants; ALK5-A (n = 30; total number of cells in gel 1,463), and ALK5-B (n = 30; total number of cells in gel 1,284). c ALK5-targeted siRNA inhibits TGFβ2-stimulated, TGFβR3-dependent ventricular endocardial cell EMT. Average percent of total GFP-expressing cells scored as epithelial, activated, or transformed. Means are derived from 3 separate experiments. All ventricular explants were given 200 pM TGFβ2. GFP served as a negative control to determine the basal level of transformation. TGFβR3 induced statistically significant increases in transformed cells with a concomitant decrease in epithelial cells. This effect was abolished in the presence of 2 independent siRNAs targeted against ALK5.d, e BMP-2-stimulated, TGFβR3-dependent ventricular endocardial cell EMT requires intact ALK5/Par6c/Smurf1 pathway signaling. Average percent of total GFP-expressing cells scored as epithelial, activated or transformed. Means are derived from 3 separate experiments. All ventricular explants were given 5 nM BMP-2. GFP served as a negative control to determine the basal level of transformation. TGFβR3 induced statistically significant increases in transformed cells with a concomitant decrease in epithelial cells. This effect was abolished in the presence of 2 independent siRNAs targeted against ALK5 (d) or 2 independent siRNAs (e) targeted to either Par6c or Smurf1. Fora, c–e, * 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, refer to online supplementary table 1.