SK-3rd TICs possess properties of EMT.

<p>A–B) Differential expression of EMT markers in SK-3rd TICs: SK-BR3 and mammosphere forming SK-3rd cells were examined by real time RT-PCR (A) and Western blotting (B) for expression of EMT markers. E-cad: E-cadherin; Vim: Vimentin; Cytok: cytokeratin. C–E) Upregulation of Twist-1 in SK-3rd TICs: The total RNA of SK-BR3 and SK-3rd cells was examined for expression of Twist-1 and Snail using real time RT-PCR (C). Housekeeping genes were used to normalize Twist-1 and Snail mRNAs in all samples. At the end of real time RT-PCR, the samples were examined in a 1.2% agarose gel (D). The cell lysates from nuclear fractions were examined by Western blotting using anti-Twist-1 antibodies (E, Upper panel). Histone 3 was used as a loading control for nuclear preparation. F) Effect of Twist-1 on MT1-MMP expression: SK-3rd TICs silenced for Twist-1 or GFP control were examined by Western blotting using antibodies as indicated for expression of MT1-MMP and Twist-1. β-actin was used as a loading control.