Fig. 3

MRP1 expression and function are induced in breast cell lines by doxorubicin in vitro. HB2 or T47D cells were treated for 24 h with 1 μM doxorubicin or vehicle control (DMSO). a MRP1 expression was quantified by qPCR (left) or Western blot (right). For qPCR, means with SD of triplicate PCR reactions are presented. Densitometry values are presented beneath MRP1 blots and pertain to the blots presented. Data for qPCR and Western blot are representative of at least 2 independent biological replicates. b Expression of canonical Notch target genes Hes1 and Hey1 was quantified by qPCR. Means are presented with SD of triplicate PCR reactions, and experiments are representative of at least 2 biological repeats. c Efflux of the fluorescent MRP1-substrate, esterified calcein, was assessed using flow-cytometry. Efflux was significantly enhanced in the presence of doxorubicin (non-linear regression T47D p = 0.0035, HB2 p = 0.0009). Analysis of individual timepoints reveal T47D cells show significantly enhanced efflux over 2-5 h post-wash out (two-tailed t-test p < 0.05 at each time point), whilst significant efflux occurred from 4 to 5 h post-wash out for HB2 cells (two-tailed t-test p < 0.01 at 4 h and p < 0.0001 at 5 h). The mean response of 4 independent biological replicates with technical triplicates is presented. Error bars represent the SEM of the 4 replicates