Fig. 4

Induction of P-gp/MDR1 expression in 17-DMAG-resistant cells. a Detection of MRPs and MDR1 mRNA was performed by quantitative RT-PCR. The sizes of the PCR products were 525 bp (MRP1), 1254 bp (MRP2), 828 bp (MRP3), 157 bp (MDR1), and 230 bp (GAPDH). b P-gp expression was assessed using western blot analysis. c Cells were treated with 1 μM Rho123 for 1 h (gray peak) and then incubated with Rho123-free media for 3 h (red blank peak). Rho123 fluorescence was analyzed by flow cytometry. Rho123 efflux was measured by counting cells at the left of the dashed line of the plot (M1 region). d Control and P-gp siRNAs (100 nM) were introduced into parental or resistant cells, and P-gp silencing was confirmed by western blot analysis. e Cells were treated with the indicated concentrations of 17-DMAG after transfection of P-gp siRNA or pretreatment with 5 μM verapamil. Cell viability was measured 72 h later using the MTT assay. f Cells were pretreated with or without 5 μM verapamil and then treated with the indicated concentrations of 17-DMAG for 6 h. The proteins involved in ALK-related signaling were detected by western blot analysis