Establishment of a Tet-regulated expression system for MCF-10A cells. (A) Schematic representation of pWHE644 and ( B ) pTET/HAhBRAF-IRES-GFP-bsr. In the absence of dox, the activator protein (yellow) displays no DNA binding activity and the repressor protein (red) binds to the expression vector, suppressing expression of the gene-of-interest. Upon dox treatment, the repressor looses, while the activator gains DNA binding activity and drives the transcription of the bi-cistronic HA-B-Raf-IRES-GFP mRNA. See Methods for further details. (C) Flow chart for the generation of Tet-inducible MCF-10A cell lines. (D) Western blot analysis for screening purposes showing the expression of both transregulator proteins in a series of independent MCF-10AecoR pools transfected with pWHE644 (MCF-10Atet). A lysate from K562 cells (Wöhrle et al, in preparation) expressing the rtTA and tTS proteins serves as positive control (K562-pWHE644). Parental MCF-10AecoR cells and mock transfected K562 cells serve as negative controls. Detection of AKT serves as loading control. Pool 19 was chosen for further evaluation and ultimately for transfection with the pTET-bsr constructs (E) Morphological comparison of MCF-10AecoR cells (a/b) and the cell pool 19 expressing the rtTA/tTS proteins (c/d) in the subconfluent state and grown to confluency, respectively. (F) Phenotypic comparison of parental MCF-10A cells  in 3D culture with three independent sublines of MCF-10Atet cells (sublines will be explained in detail below) at day 15. Note that all four sublines form acini displaying ongoing luminal clearance as indicated by the high number of cells stained with an antibody for cleaved caspase-3.