Fig. 6. Involvement of multi-drug resistance transporters in CFZ sensitivity.

a Functional inhibition of ABCB1 (PgP) multi-drug transporters by lopinavir and reserpine in 4 studied cell lines and patient-derived primary cells. The data represent the mean MFI of MTG ± SD from 3 independent experiments for cell lines and the single value for patient-derived primary samples. Statistical significance was determined with unpaired t-test, ** represents p < 0.01; *** represents p < 0.001. b Correlation between fold change of MFI of MTG by lopinavir and fold change of IC₅₀ of carfilzomib by lopinavir. The data represent the correlation between the mean values from three independent experiments from cell lines and single values from patient-derived primary cells. The p-value and correlation coefficient (r) were determined by Pearson correlation. c Representative western blot image of ABCB1 level in MDA-MB-231 cells stably transduced with ABCB1 protein (PgP+). GAPDH serves as a protein loading control. d Functional inhibition of ABCB1 by lopinavir and reserpine in PgP+ and PgP- MDA-MB-231 cells. The data represent the mean MFI of MTG ± SD from 3 independent experiments. Statistical significance was determined with unpaired t-test, ** represents p < 0.01; *** represents p < 0.001. Dose-response curves of MDA-MB-231 cells without PgP (PgP-) or with introduced PgP (PgP + ) to bortezomib (e) and carfilzomib (f) in monotherapy or in combination with lopinavir. The cells were treated with PIs for 1 h and subsequently placed into a drug-free medium or into the medium with lopinavir for 48 h. The data represent the mean ± SD from 3 independent experiments. g Estimation of ABCG2 in 4 studied cell lines on the mRNA level by qPCR (upper part) and on the protein level (lower part) by western blot. In both experiments, the levels were normalized to GAPDH, which served as a control. h Representative western blot image of ABCG2 knock-out in control or in two single-cell derived clones #3 and #5 of MCF-7 cell line. i Cytotoxicity of carfilzomib in MCF-7 cells with a normal level of ABCG2 (NC) or in single-cell derived clones (#3 and #5) with knocked-out ABCG2. Viability was assessed after 1 h pulse treatment with carfilzomib and continuous 48 h incubation in a drug-free medium. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired t-test. j Cytotoxicity of carfilzomib and lopinavir combination in MCF-7 cells with a normal level of ABCG2 (NC) or in single-cell derived clones (#3 and #5) with knocked-out ABCG2. Viability was assessed after 1 h pulse treatment with carfilzomib and continuous 48 h treatment with lopinavir. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired t-test. ABCB1 ATP Binding Cassette Subfamily B Member 1, ABCG2 ATP Binding Cassette Subfamily G Member 2, BTZ bortezomib, CFZ carfilzomib, GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase, LPV lopinavir, MFI median fluorescence intensity, MTG MitoTracker Green FM, PgP P-glycoprotein (ABCB1).