Fig 2: Expression and activity of ABCCs/MRPs. Panel (A): The apical-to-basolateral (AB) and basolateral-to-apical (BA) fluxes of 0.5 µM 3H-estrone-3-sulfate were monitored both in the absence (none) and in the presence of 50 µM MK571, as indicated. Data obtained were employed to calculate the efflux ratio (ER) and uptake ratio (UR), as defined in Materials and Methods. Bars are the mean ± SEM of 3 independent determinations (single dots). * p < 0.05 vs. none with a two-tailed Student’s t-test. Panel (B): The expression of ABCC1/MRP1 and ABCC2/MRP2 genes and proteins were performed with RT-qPCR or and Western blot analyses, respectively, as described in Materials and Methods. Data of mRNA expression are the mean ± SEM of 3 independent determinations (single dots), while representative Western blots are shown that, repeated twice, gave comparable results. Panel (C): Immunolocalization of MRP1 in hAELVi and EpiAlveolar™. Confocal laser scanning microscopy of monolayers immunolabeled for MRP1 (green) is shown. Nuclei were stained with propidium iodide (red). Top, single XY scans of hAELVi (left) and EpiAlveolar™ (right) are shown; XZ sections of the planes are also shown in the rectangle images. Bottom, tridimensional reconstruction of acquired horizontal sections. Representative images from three independent experiments are shown. Bar = 20 µm.

Fig 3: Efflux pumps and their involvement in the intracellular availability of carfilzomib in NSCLC cell lines. (A) Expression of ATP-type transporters ABCB1, ABCC2 and ABCG2 in A549, H157, H460 and H1703 cell lines as determined by quantitative real-time PCR. Data are normalized to GAPDH, expressed in 2−dCt values and are presented as the mean ± SD of at least 3 replicates. (B) Levels of ATP-type transporters ABCC2 and ABCG2 in the same cell lines determined by western blot. Data show a representative blot of 3 replicates. (C, D) Active site labeling with proteasome subunit-selective probes was performed after (C) bortezomib or (D) carfilzomib pulse treatment (both drugs 10 nM and 20 nM) alone or in combination with nelfinavir (20 µM for A549; 10 µM for H460, H157 and H1703) for 1 h in NSCLC cell lines. Proteasome subunit activities are illustrated in different colors as follows: β1 in blue, β2 in green and β5 in red. Data represent the mean ± SD of at least two replicates. The original data are presented in Fig. S3. * Represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

Fig 4: Effects of different theaflavin monomers on the mRNA and protein levels of MRP1, MRP2, MRP3, P-gp and BCRP in Caco-2 cells. (A) Effect of theaflavins on mRNA levels of efflux transporters. (B) Effect of theaflavins on protein levels of efflux transporters. Ctrl: Caco-2 cells were incubated with HBSS; TF, TF3G, TF3’G and TFDG: Caco-2 cells were incubated with TF, TF3G, TF3’G and TFDG. Results are expressed as means ± SD (N = 3). Different small letters indicate significant difference between different treatments at p<.05.

Fig 5: Multidrug resistance (MDR) assay in CML-T1 and CML-T1/IR cells and immunodetection of MRP2 protein by western blot analysis in the CML-T1 and CML-T1/IR cells compared with other cell lysates. (A) Measurement of calcein retention as a surrogate of MDR exporter activity in the CML-T1 and CML-T1/IR cells was performed as described in 'Materials and methods'. Cyclosporine A (CsA) and verapamil were used as inhibitors of MDR. No MDR activity was detected in the CMLT1 and CMLT1/IR cells as reflected by 100% calcein retention in the cells. (B) Cell lysates of CML-T1 and CML-T1/IR were subjected to SDS-PAGE and western blot analysis for immunodetection of MRP2 together with the lysates of K562, HL-60, HeLa cells and mouse liver tissue, which were used as MRP2 positive controls.