Figure 3

S1P ₁ R mediates cell rounding, cellular outgrowth, and EMT in AV canal cultures. AV canals were explanted at E9.5 and treated at 2 and 24 hours with vehicle or S1P receptor agonists or antagonists. Explants were stained with phalloidin conjugated to Alexa Fluor 488. (A) Vehicle treated explants show normal cellular outgrowth and elongated cell morphology. (B) S1P (2.5 μM) treatment causes reduced outgrowth and cell rounding. (C) CAY10444 (2.5 μM), an S1P₃R antagonist, treated cultures have normal cellular outgrowth and cell morphology. (D) CAY10444 (2.5 μM) is unable to rescue explants from S1P (2.5 μM) treatment, as these cultures have inhibited cell migration and rounded cells. (E) The S1P₁R specific agonist, KRP203 (0.5 μM) causes reduced cellular outgrowth and cell rounding similar to S1P treatment. (F) FTY720 (1.0 μM), an S1P_1,3,4,5R agonist also causes reduced cellular outgrowth and cell rounding similar to S1P. (G) The average cellular outgrowth for each treatment was calculated and displayed as a percent of the vehicle control cellular outgrowth. (H) Each explant was examined for transformed mesenchymal cells that had invaded into the collagen gel. An explant was scored as inhibited if less than 5 mesenchymal cells had invaded. Results are displayed as a percent of explants that were inhibited. Vehicle control and CAY10444 showed no inhibition, while the other treatments had significant inhibition of mesenchymal cell formation. Scale bar low magnification equals 200 μm, and in high magnification inserts it equals 50 μm. N equals number of explants measured. *P ≤ 6.3 × 10^-5, **P ≤ 0.0005.