Figure 2.

Attenuation of MEK activity results in inhibition of MMP9 expression and phenotypic reversion of the malignant T4-2 cells. (A) Immunoblots of total cell lysates from T4-2^vector cells treated with indicated inhibitors for 7 d in 3D lrECM cultures. Phenotypic reversion of T4-2 cells is accompanied by decreased signaling through MEK. Ponceau-stained bands were used as a loading control. (B) Immunoblot of total cell lysates from T4-2^GFP cells transduced with control shGFP or with shRNAs against MEK1 and MEK2. Total level and the phosphorylated form of these two proteins are only slightly reduced in 2D cultures, but are significantly reduced in 3D cultures. Ponceau-stained bands were used as a loading control. (C) Cumulative population doublings obtained from T4-2^GFP cells transduced with shGFP or shMEK1/2. Down-modulation of MEK1/2 does not significantly change proliferation in 2D cultures. (D) shGFP- and shMEK1/2-transduced T4-2^GFP cells maintained in 3D lrECM for 7 d. Down-modulation of shMEK1/2 restores basal polarity. (Red) Nucleus; (green) α6-integrin. (E) Zymography of CM from 3D lrECM of shGFP-transduced (left) and shMEK1/2-transduced (right) T4-2^GFP cells. MMP9 production is lost with reversion induced by down-modulation of MEK1/2.