Effect of calcium on TGF-β1–mediated signaling and β-catenin–p-Smad2 complex formation. (A) Phase photographs of α3 wt cells cultured in either low (125 µM) or high (1.8 mM) calcium media showing cells cultured in high calcium media form tight cell–cell contact, whereas cells cultured in low calcium media have loose cell–cell contact. Bar, 50 µm. (B) Confocal microscopy of E-cadherin clusters in α3 wt cells cultured under either low (125 µM) or high (1.8 mM) calcium conditions. α3 wt cells cultured in high calcium media show that E-cadherin clusters occur mainly in cells at the periphery of the colony, whereas α3 wt cells cultured in low calcium media form E-cadherin clusters throughout the colony. The boxed areas are shown in higher magnifications in the bottom panels. Bar, 2 µm. (C) Serum-starved α3 wt and H245A mutant cells under high or low calcium conditions were stimulated with TGF-β1 for 48 h. Supernatants were removed from the plates and concentrated for zymography. α3 wt cells cultured in low calcium show higher MMP-9 levels than α3 wt cells cultured in high calcium after TGF-β1 stimulation. (D) Serum-starved α3 wt cells in high or low calcium media were stimulated with TGF-β1 for 48 h, and the lysates were blotted for α-SMA. α-SMA is only up-regulated in cells cultured in low calcium medium and not in high calcium condition. (E) Cells cultured under low calcium condition show increased β-catenin–p-Smad2 complex formation. α3 wt cells cultured in either high or low calcium medium were treated with TGF-β1, and the lysates were subjected to β-catenin IP followed by p-Smad2 immunoblotting. The β-catenin–p-Smad2 complex formation is only seen with cells cultured in low calcium condition and not in high calcium condition. All of the aforementioned experiments have been performed at least three times with similar results.