Figure 7.

Downregulation of EP300 or CDH1 leads to doxorubicin resistance and a phenotype characteristic of EMT. MTMEC cells were stably transfected to express small hairpins targeting EP300 and CDH1. (a) Knock-down efficiency was monitored by reverse transcription and real time PCR (upper panels) and immunoblotting (lower panels). (b) Downregulation of EP300 or CDH1 increases the motility and invasion of MTMEC cells. Upper panels, wound-healing assay; lower panel, solubilization of crystal violet to quantify cells in the transwells with (invasion) or without (migration) Matrigel (left panels). (c and d) Downregulation of EP300 or CDH1 bypasses doxorubicin-induced senescence and enables the generation of proliferating, doxorubicin-resistant clones. (c) Cells were treated with 60 ng/ml doxorubicin for 24 h and cells stained for SA-β-galactosidase 3 weeks after. (d) Cells were treated for 24 h with 60 ng/ml doxorubicin and drug-resistant clones were stained with crystal violet after 3 weeks (left panels) and counted (right panels). (e) Cells were treated with doxorubicin as above and 3 weeks later, trypsinized and seeded (10 000 cells per well in a 96-well plate). Percentage of cells incorporating EdU by fluorescence microscopy (left panels) and EdU incorporation determined by fluorescence detection (right panel). (f) Anchorage independence was determined by the formation of clones in soft agar after 4 weeks. Numerical data represent the average±S.D. of at least three different experiments (*P<0.05). Pictorial data were repeated at least in triplicate and a representative picture is shown