Figure 5.

Reducing endogenous miR-200 expression induces EMT in HCT116 cells. (A) Cells were transfected repeatedly every 3 d with either scrambled LNA (S), LNA-200, or LNA-let-7 oligo. Expression of mRNAs was quantified by real-time PCR 18 d after the first transfection. (B) Immunofluorescence analysis of E-cadherin expression (pink) in cells repeatedly transfected for 22 d. Cells were counterstained with DAPI. (C) Western blot analysis of cells treated for 15 d. (D) Phase contrast picture showing cells treated for 15 d. (E) Cells were first transfected with either 50 nM LNA scrambled or LNA-200, and after 24 h, once E-cadherin was up-regulated, they were again transfected with either scrambled siRNA pool, a ZEB1-specific siRNA pool, a ZEB2-specific siRNA pool, or both. After 72 h, expression of ZEB1, ZEB2, and E-cadherin was quantified by real-time PCR. (F) Cells were transfected with empty vector (vec) or with HA-ZEB2, and after 3 d expression of E-cadherin, miR-200a, and miR-200c was quantified by real-time PCR. Numbers were normalized to a transfection efficiency of 48%. Expression of HA-ZEB2 was confirmed by Western blot analysis (not shown).