Intracellular transport study. Caco-2
BYL719 were grown as epithelial layers by seeding them at a density of 2 × 105 cells/well on polycarbonate Transwell? filters (Corning, Schiphol-Rijk, The Netherlands) and studies were performed 14 days after plating. Afterwards cells were transfected with SiQD–siRNA or Lipofectamine–siRNA. To ensure cell monolayer integrity the transepithelial electrical resistance (TEER) was measured before and after each experiment using the Millicell–ERS system (Millipore, Schwalbach, Germany). Rhodamine 123 (Rh123) dissolved at 0.1 mM in serum and aminoacid-free B2 buffer. This buffer was used because of the fluorescent substances in DMEM and Optimem and its high cell viability. The Rh123 solution was added to the donor compartment and the Rh123 transport in secretory direction was monitored by measuring the fluorescence intensity in the acceptor compartment as function of time using a home-made in situ fluorescence detector device [14]. The functional changes in the efflux due to ABCB1 knockdown was measured over a
period of 10 days performing a transport study each day for 5 h.
