Fig. 3

Lactate confers a potent chemoresistance through upregulation of MRP1. a Hoechst 33342 efflux assays of cells treated with 20 mM LA in the presence and absence of ABC transporter inhibitor 10 μM Verapamil using BioTek citation 5 (BioTek, Winooski, VT). Representative histogram plots are shown of non-treated cells (control), 20 mM LA, 10 μM Verapamil and combined treatment of lactate and Verapamil. The result clearly showed that lactate significantly promoted the efflux of Hoechst 33342 in A549 and H1299 cells (**P<0.01, ***p<0.001 for difference from untreated control, ^###p<0.001 for difference from LA-treated cells by ANOVA with Dunnett’s correction for multiple comparisons). b Quantitative real-time PCR was performed for analysis of mRNA expression levels of several members of ABC transporter family. Values represent the relative increase of MRP1 mRNA normalized to GAPDH. (***p<0.001 for difference from untreated control by ANOVA for multiple comparison). c A549 cells were co-transfected with MRP1 promoter reporter construct and control Renilla luciferase reporter plasmid and treated with indicated lactate concentrations after 48 h. Luciferase activity was determined and normalized using the dual luciferase reporter system (**P<0.01, ***p<0.001 for difference from untreated control by ANOVA for multiple comparison). d Western blot demonstrates increased MRP1 expression following 3 h of LA (0, 5, 10, 15, 20 and 30 mM) stimulation in both A549 and H1299 cells. e 48 h after transfection of A549 either with MRP1 siRNAs or control siRNA, and then cells were stimulated first with LA for 3 h and further etoposide for additional 36 h before western blotting. The results showed that the apoptosis marker Cleaved-PARP was significantly induced after knockdown of MRP1. f A549 cells were stimulation with different doses of LA for 3 h and then further exposed to indicated drugs for additional 36 h, western blot results show that LA can significantly reduce Cisplatin, Doxorubicin-induced levels of Cleaved-PARP and γ-H2AX. g Cell viability assays show that LA can significantly increase A549 cells survival in the presence of cisplatin (30 mM) and doxorubicin (30 mM). h The cells were treated as described in Fig. 3f, western blot results show that LA can significantly reduce Taxol-induced levels of Cleaved-PARP, but not that of γ-H2AX. i Cell viability assays show that lactate can significantly increase A549 cells survival in the presence of Taxol (25 mM)