Fig. 2.

NUMB6 overexpression induces EMT-associated alterations in DB-7 cells. (A) Alterations of epithelial and mesenchymal markers expression in NUMB6-GFP DB-7 cells. Overexpression of NUMB6 reduces E-cadherin and increases expression of mesenchymal markers such as vimentin, fibronectin, and FSP-1, active β-catenin, Slug and MMP9. Protein lysates from DB-7 cells that stably expressed NUMB4-GFP, NUMB6-GFP, or vector alone with GFP were analyzed by immunoblot with antibodies against E-cadherin, fibronectin, vimentin, and FSP-1, active β-catenin, total β-catenin, Slug MMP-9 and β-actin. (B) Immunofluorescence study showing NUMB6 regulation of the expression and localization of EMT markers. NUMB4-GFP, NUMB6-GFP and GFP DB-7 cells were immunostained with E-cadherin, active β-catenin, FSP-1, vimentin, or Slug antibodies. Red is Alexa Fluor 594 anti-rabbit or anti-mouse. Nuclei were stained with DAPI. Bars=20um. (C) Numb6 regulation of E-cadherin transcription. NUMB6 caused loss of E-cadherin mRNA whereas NUMB4 increased it. The mRNA levels of E-cadherin were measured by quantitative RT-PCR analysis of total RNA extracted from NUMB4-GFP, NUMB6-GFP or control vector GFP DB-7 cells. The mRNA levels of E-cadherin are expressed relative to β-actin transcripts. Each experiment was performed in triplicate and repeated three times. Error bars represent standard error of the mean (SEM). (D) NUMB6 overexpression downregulated E-cadherin luciferase reporter activity. NUMB4-GFP, NUMB6-GFP or control vector GFP DB-7 cells were transfected with the E-cadherin-LUC reporter vector and Renilla constructs for 24 h before measuring luciferase activity. Cells were harvested after 24 h and lysates were assayed for luciferase activity. The relative luciferase activities are expressed in arbitrary units, and were normalized to co-transfected Renilla activity to control for transfection efficiency. Bars represent fold of change over control GFP DB7 cells. Each experiment was performed in triplicates. Error bars represent SEM. (E) NUMB6 overexpression induces Slug expression. The mRNA levels of Slug were measured by quantitative RT-PCR analysis of RNA extracted from DB-7 cells stably expressing NUMB4-GFP, NUMB6-GFP, or control vector GFP. The mRNA levels of Slug are expressed relative to β-actin transcripts. Each experiment was performed in triplicate. Error bars represent SEM.