Figure 3

Conditional Tet-regulated transgene expression in 3D cultures of MCF-10A cells. (A) Overview on acinar development based on [1, 7]. At day (d) 0, single MCF-10A cells are seeded into matrigel. Apico-basal polarization and luminal cell death occur after one week in culture. Two weeks later, hollow acini are surrounded by a thick, mostly single cell layer epithelium. During the third week, proliferation ceases, while the epithelium becomes thinner. (B) Expression of B-Raf^V600E in mid-term acini perturbs their morphogenesis. At d10, one culture was continued in the absence of dox, while the other was grown in the presence of dox for 14 days. (C). The basal lamina of MCF-10A acini is disrupted by cells expressing oncogenic B-Raf^V600E. Thirty-three days old acini grown in the presence of dox for 17 days were fixed and stained with an antibody for laminin V. Note the diffuse laminin V deposition surrounding cells expressing B-Raf^V600E (indicated by an arrow). (D) Dox withdrawal leads to a loss of invasive structures and reappearance of E-Cadherin. Three D cultures of MCF-10A cells harboring a dox-inducible B-Raf^V600E oncogene were seeded and switched to dox-containing medium at day 17 for 18 days. Then, dox was withdrawn from one culture (dox wash-out), while the other culture was kept under dox. Following another 24 days, the cultures were fixed and stained with anti-E-Cadherin antibodies (red) and Topro-3 (blue) to identify cell-cell contacts and nuclei, respectively. (E) Western blot analysis of 3D cultures showing tight regulation of the rtTA2^S-M2/tTS^D-PP expression system. MCF-10A cells harboring the inducible B-Raf^V600E or empty vector control constructs were induced at day 6 after seeding and harvested 4 days later.