Figure 4

Comparison of BCRP protein and mRNA expression and stability in LTLTCa and MCF-7Ca cells. LTLTCa and parental MCF-7Ca cells were plated and cultured in their respective passage media under normal cell culture (nonhypoxic) conditions. A) Total protein was extracted and BCRP and β-actin were analyzed by Western blot analysis. Densitometry results are expressed as fold-change in protein levels compared to MCF-7Ca cells after normalization to β-actin (mean ± SD, n = 6 independent cell samples/group; *versus MCF-7Ca, P <0.0001, two-sided t test). B) Total RNA was extracted and BCRP mRNA, VEGF mRNA and 18S rRNA were analyzed by real-time RT-PCR analysis. Results are expressed as the fold-change in mRNA levels compared with MCF-7Ca cells after normalization to 18S rRNA (mean ± SD, n = 6 independent cell samples/group; *versus MCF-7Ca, P <0.0001, two-tailed t-test). C) LTLTCa and MCF-7Ca cells were treated with vehicle or 0.5 μg/ml actinomycin D for 0 to 16 hours. Total RNA was extracted and BCRP mRNA was analyzed by real-time RT-PCR. Results are expressed as least square means of log transformed averages of mRNA expression at various timepoints (trend over time) after normalization to corresponding vehicle-treated samples, and analysis by linear mixed effect model adjusting for experiment, cell line, and cell line*time interaction (mean ± SD of n = 6 independent cell samples/group; *versus MCF-7Ca; effect of gene type P = 0.0025, effect of cell type P = .3749, interaction between gene type and cell type P = 0.0025; two-way ANOVA). ANOVA, analysis of variance; BCRP, breast cancer resistant protein; n, number; SD, standard deviation; VEGF, vascular endothelial growth factor.