Figure 6. Wnt1/3a promote L cell elongation via a ß-catenin independent pathway.

Panel A: Mock-transfected L cells were treated with control-conditioned media (Control-CM) or Wnt1/3a-conditioned media for 24 hrs. Note that Wnt1-CM was produced by standard L cells while Wnt3a was produced by E-cadherin transfected L cells (EL cells). Cells were then fixed, immunostained for PDI, and imaged with a confocal microscope. The length of the long axis of cells was measured using Adobe Photoshop (version 9.0.2). Error bars represent standard error. This experiment was repeated twice. At least 100 cells in 5 fields were measured. A Student's t-test shows that the difference in cell length between cells treated with control-CM and Wnt1- or Wnt3a-CM is statistically significant (p<0.005). Panel B: LS/L cells were treated with control-CM from mock-transfected L cells, Wnt1 conditioned medium, 33 mM NaCl, or 33 mM LiCl for 20–30 hrs and then assayed for activation of the SuperTopFlash reporter. Panel C: L cells were treated with 33 mM NaCl or LiCl for 24 hrs and then analyzed for cell elongation as in Panel A. Error bars represent the standard error from over 150 cells in two independent experiments. Neither NaCl nor LiCl has any significant effect on the length of the cells. Panel D: Purified mWnt3a was added to LS/L cells 24 hrs prior to conducting a dual luciferase assay to measure the activation of the SuperTopFlash construct. Error bars represent the standard error from 2 independent replicates with a total of 8 data points. Panel E: Mock-transfected L cells were incubated with purified mWnt3a for 24 hrs. Cells were then fixed, immunostained with anti-PDI, and imaged via confocal microscopy. The length of the long axis of individual cells was measured in Adobe Photoshop. Error bars represent the standard error from 2 independent replicates with a minimum of 107 cells measured for each data point.