Fig 2: (A) The expression of ABCB1 and GAPDH in SW620/Ad300 cells after treatment with 1 µM tetrandrine at 0, 24, 48, and 72 h time points. (B) representation of ABCB1/GAPDH relative ratio of A; (C) The expression of ABCB1 and GAPDH in KB-C2 cells after treatment with 1 µM tetrandrine at 0, 24, 48, and 72 h time points. (D) representation of ABCB1/GAPDH relative ratio of C; (E) Immunofluorescent staining on the influence of tetrandrine on the subcellular localization of ABCB1 in SW620/Ad300 cells at 0, 24, 48, and 72 h time points. The nuclei were stained by PI (Propidium Iodide) and shown in red.

Fig 3: 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

Fig 4: Intracellular distribution of doxorubicin in RBE4 wildtype and -MDR1-EGFP cocultures. RBE4 wildtype and –MDR1-EGFP cocultures were stained for endolysosomal vesicles with LysoTracker (blue) and treated with the Pgp substrate doxorubicin (red) for 30 min, before live-cell imaging using a confocal laser scanning microscope. Insets show a magnification of endolysosomal localization of doxorubicin and Pgp (green). In RBE4 wildtype cells, doxorubicin binds to its nuclear target as visible by red staining (#1), while nuclei of Pgp-EGFP overexpressing cells lack a nuclear doxorubicin staining (#2). Similar experiments with similar outcomes were previously performed with cocultures of wildtype and MDR1-EGFP-transduced hCMEC/D3 cells [31], and therefore these experiments were not repeated and again illustrated here

Fig 5: Antibody and pravastatin binding on MDR1-incorporated tBLM by SPR. (A) (?) Binding result of anti-MDR1 monoclonal antibody on MDR1-incorporated tBLM. (¦) Binding result of anti-MDR1 antibody on MDR1-free tBLM. (?) Binding result of anti-Pin-1 (G8) mouse monoclonal antibody on MDR1-incorporated tBLM. (B) Binding result of (?) 0.05 mg/mL and (?) 0.01 mg/mL pravastatin solutions on MDR1-incorporated tBLM and binding of 0.05 mg/mL of pravastatin (?) on MDR1-free bilayers.