Figure 2
Functional evaluation of DvABCB1 by heterologous expression in HEK293 cells and quantitation of cell toxicity to IP3-H9. (a) Dose-dependent morphology change of HEK293 cells expressing Dv-ABCB1 after 24 h of IP3-H9 treatment. HEK293 cells were transfected with DvABCB1 vector and selected with 1 mg/mL of Geneticin for 4 weeks before treated with 0–100 nM of IP3-H9. Expression of DvABCB1-RFP fusion protein was shown as an indicator of DvABCB1 positive cells (left panel, 0 nM IP3-H9 as a representative). (b) Cytotoxicity of IP3-H9 to the HEK293 cells expressing DvABCB1. Cells were seeded in 96-well plates overnight before cultured with 0, 0.01, 0.1, 0.5, 1, 5, 10 and 100 nM of IP3-H9 for 24 h. The ATP level of metabolically active cells were quantified by the CellTiter-Glo Viability assay. The luminescence of 10 µM oligomycin challenged cells was subtracted from that of the toxin treated cells as the background absorbance. The cell viability was normalized by using 0 nM IP3-H9 treated cells as 100%. n = 3. (c) DvABCB1-RFP (red) was expressed on the cellular surface of HEK293 cells. Cell nuclei were stained with DAPI (blue).
