Figure 5. Activation of STAT5 induces molecular and phenotypic changes consistent with EMT.
(A) Activation of STAT5 abrogates E-cadherin expression and increases vimentin expression in SCCHN cells. PCI-15B pUSEamp #1 cells and cells from two representative STAT5 dominant-active clones; PCI-15B STAT5a 1*6 #2 and PCI-15B STAT5b 1*6 #15, were harvested for Western blot analysis. Expression of E-cadherin was totally abrogated in the STAT5 constitutive-active cells when compared with the PCI-15B pUSEamp #1 control. The loss of E-cadherin was accompanied by an increase in vimentin expression. Actin is shown as loading control. (B) Activation of STAT5 results in cell scattering and reduced cell-cell adhesion in SCCHN cells. The stable clones, PCI-15B pUSEamp #1 and PCI-15B STAT5a 1*6 #2 were plated (3×105 cells, each) in tissue culture dishes. Twenty-four hours after plating, cellular morphology was recorded by digital camera under a microscope (at 40X and 200X magnification). The experiment was performed twice with similar results. (C) 3 × 105 cells of of PCI-15B cells were plated in triplicates in 6 well plates. 24 hours after plating, cells were transiently co-transfected with either 1.5 μg of E-cadherin Luc and 1.5 μg of pUSE amp (+) or 1.5 μg of E-cadherin Luc and 1.5 μg of pSTAT5a DA plasmids. Luciferase assay was performed 48 hours after transfection. Luciferase activity was expressed as relative light units per microgram of total protein (RLU/g protein). RLU/μg values from vector control and E-cadherin-Luc transfected cells were normalized to 100%, and decrease in RLU/μg of pSTAT5a DA and E-cadherin Luc transfected cells was calculated (p=0.002). P values were calculated from several independent experiments, performed in triplicate each time.