Figure 2.

Effects of ensartinib on the intracellular accumulation of hoechst 33342 (A,B), calcein AM (C), daunorubicin (D,F) and mitoxantrone (E) in MDCKII cells transduced with the human ATP-binding cassette (ABC) transporters ABCB1 (A,D), ABCG2 (B,E), and ABCC1 (C,F). In hoechst 3342 and calcein AM assays, cells were pre-incubated with ensartinib at various concentrations for 10 min. The substrates (8 µM hoechst 3342 or 2 µM calcein AM) were then added and their accumulation in intact cells was monitored directly using a fluorimeter in kinetic mode. The endpoint interval was used for data evaluation. In daunorubicin and mitoxantrone assays, cells were pre-incubated with ensartinib or model inhibitors for 10 min, then treated with 2 µM daunorubicin or 1 µM mitoxantrone, respectively. After 1 h incubation, the cells were trypsinized and the substrates’ fluorescence was measured with a flow cytometer. In both cases, LY335979 (1 µM), Ko143 (1 µM), and MK571 (25 µM) were used as model ABCB1, ABCG2, and ABCC1 inhibitors, respectively. These inhibitor concentrations induced maximal inhibition of the transporters, so the results obtained with the model inhibitors were taken to represent 100% inhibition when calculating absolute IC₅₀ values for ensartinib. The plotted relative accumulation values represent fold-increase in probe substrate accumulation elicited by tested compound and are expressed as ratios of relative fluorescence units (RFUs) from treated samples to RFUs of control in particular cell subline. The data are means ± SD based on three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 relative to control).