Fig. 11

Increased number and size of intracellular Pgp-EGFP-positive vesicles after treatment of RBE4-MDR1-EGFP or hCMEC/D3-MDR1-EGFP cells with doxorubicin (DOXO). Comparison of the number and size of intracellular Pgp-EGFP positive vesicles in RBE4- and hCMEC/D3-MDR1-EGFP cells before (control) and 24 h after treatment with DOXO (10 µM). Live-cell image acquisition was performed with an inverted fluorescence Lumascope 620 in the cell culture incubator. In both cell lines, Pgp-EGFP (green fluorescent) was localized in the cell membrane (orange arrows), whereas Pgp-positive intracellular vesicles (white arrows), most likely of endolysosomal origin, were seen in hCMEC/D3 but hardly in RBE4 cells (similar to the confocal laser scanning photomicrographs shown in Fig. 1). Exposure to DOXO increased the number of Pgp-EGFP positive vesicles in both cell lines. Furthermore, the size of the Pgp-EGFP positive vesicles appeared larger after DOXO exposure (white boxes). An increase of the number of intracellular green-fluorescent vesicles after DOXO treatment may indicate biogenesis of Pgp-containing intracellular vesicles and/or transport of Pgp into the vesicles. Those mechanisms likely enhance drug sequestration capacity of the cells after exposure to cytotoxic compounds/Pgp-substrates. See Additional file 5 for quantification of Pgp-EGFP positive vesicles before and after treatment with DOXO