Fig 2: RNA and protein expression of selected ABC transporters, which are known to be involved in taxane resistance and/or MDR, in DU145 and PC-3 DTXR cells compared to CTRL cells. (a) The relative transcript levels of eight selected ABC transporters were plotted on a heat map. Cells with an expression of the respective ABC transporter beyond the detection threshold (CP ≥ 35) are marked with an asterisk (*). (b) Exemplary Western blots for detecting ABCB1 and ABCG2 proteins in CTRL and DTXR cells. GAPDH served as a reference protein. (c,d) Exemplary flow cytometry analysis for (c) ABCB1 and (d) ABCG2 proteins via specific antibodies conjugated with the fluorescent phycoerythrin (PE) in CTRL and DTXR cells. The vertical black lines indicate the threshold of unspecificity according to the pooled isotype controls. The adjacent graphs to the right depict the median fluorescence intensities (MFI) for ABCB1 and ABCG2 protein, respectively. Data are depicted as mean ± standard deviation (SD) of three to four independent biological replicates. Non-paired t-test with Welch’s correction was performed for comparison of CTRL vs. DTXR cells: *** p < 0.001, ns: not significant.

Fig 3: Influence of siRNA-mediated knockdown of ABCB1 in combination with DTX on cellular viability and clonogenic survival of DU145 and PC-3 DTXR cells. (a) The relative transcript and protein levels of ABCB1 in DTXR cells 96 h after siRNA transfection start are depicted. An exemplary Western blot for the detection of ABCB1 protein with GAPDH as reference protein is shown. (b) DU145 and PC-3 DTXR cells were treated with a serial dilution of DTX after transfection with either siR-CON or siR-ABCB1 (20 nM). After treatment, the metabolic activity was measured by the WST-1 assay and IC50 values for DTX were calculated. The adjacent graphs to the right depict IC50 values calculated from individual experiments. (c) DTXR cells were transfected with either siR-CON or siR-ABCB1 (20 nM) and 96 h after transfection start the cells were treated with 50 nM DTX. Thereafter, cell colony formation assays were seeded and cell colonies were counted after 10–12 d. Exemplary colonies are shown in the right panels. All data were normalized to the indicated control (black line). Data are depicted as mean ± SD of three to five independent biological replicates. (a,c) One-sample t-test for comparison to the siR-CON-treated controls and (b,c) non-paired t-test with Welch’s correction for comparison of two treatment groups: * p < 0.05, *** p < 0.001, ns: not significant.

Fig 4: Influence of the glycosylation status of ABCB1 protein on ABCB1 efflux activity of the fluorescent ABCB1 substrate Rh123 in DU145 and PC-3 DTXR cells. (a) DU145 and (b) PC-3 DTXR cells with or without pre-treatment with 100 ng/mL tunicamycin (TUN) for 96 h were incubated with 0.5 µg/mL Rh123 for 15 min. After several washing steps, RPMI 1640 medium was added and subsequently the cells were imaged in 10 min intervals. As examples, the first image (t0) and images taken after 10 and 20 min are depicted for DTXR cells with or without tunicamycin pre-treatment (scale bar 50 µm). Longitudinal data were normalized to DTXR cells without tunicamycin pre-treatment at t0 (black line in the graph). Data are depicted as mean ± SD of four to five independent biological replicates. Non-paired t-test with Welch’s correction was performed for comparison of DTXR cells with tunicamycin pre-treatment vs. DTXR cells without tunicamycin pre-treatment at each timepoint: * p < 0.05, ** p < 0.01, ns: not significant.

Fig 5: Influence of the ABCB1 inhibitors elacridar and tariquidar in combination with DTX on cellular viability and clonogenic survival of DU145 and PC-3 DTXR cells compared to CTRL cells. (a) DU145 and PC-3 CTRL and DTXR cells were treated with a serial dilution of DTX in the presence or absence of an ABCB1 inhibitor (50 nM elacridar or tariquidar). After treatment, the metabolic activity was measured by the WST-1 assay and IC50 values for DTX were calculated. The adjacent graphs to the right depict IC50 values calculated from individual experiments. Ela.: elacridar, Tari.: tariquidar. (b) CTRL and DTXR cells were treated with either 50 nM elacridar or 10 nM DTX alone or with a combination of both. Thereafter, cell colony formation assays were seeded and cell colonies were counted after 10–12 d. Exemplary colonies are shown in the right panels. All data were normalized to the indicated control (black line). Data are depicted as mean ± SD of four to six independent biological replicates. (a) Non-paired t-test with Welch’s correction for comparison of two treatment groups and (b) one-sample t-test for comparison to the untreated control: ** p < 0.01, *** p < 0.001, ns: not significant.