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. 2012 Feb;40(2):304–312. doi: 10.1124/dmd.111.042721

Fig. 5.

Fig. 5.

Both Pgp and ABCG2 in are functionally expressed in HeLa cells cotransduced with BacMam-Pgp and BacMam-ABCG2 viruses. a and b, HeLa cells were cotransduced with BacMam-Pgp and BacMam-ABCG2 (1:40 virus titer for Pgp and 1:60 virus titer for ABCG2) alone or together, as described under Materials and Methods. The function of Pgp (a) and ABCG2 (b) in single- or double-transduced cells (as indicated on histograms) was evaluated by calcein-AM accumulation assay in the presence or absence of 2 μM XR9576 (for Pgp) and pheophorbide A accumulation assay in the presence or absence of 5 μM FTC (for ABCG2) as described under Materials and Methods. The histograms represent fluorescence intensity (x-axis) plotted as a function of cell number (y-axis) and are representative of three independent experiments. c, determination of contribution of Pgp and ABCG2 in efflux of mitoxantrone from HeLa cells transduced with both Pgp and ABCG2 BacMam virus: HeLa cells were transduced with BacMam-Pgp, BacMam-ABCG2, or both viruses to express/coexpress Pgp, ABCG2, or both Pgp and ABCG2 simultaneously. The Pgp and ABCG2 transport function was detected by incubating cells with 5 μM mitoxantrone for 45 min at 37°C in the dark in the absence or presence of 20 μM vinblastine (as an inhibitor of Pgp transport) and/or 5 μM FTC (as an inhibitor of ABCG2 function). The cells were then washed and were subsequently analyzed by flow cytometry as described under Materials and Methods. The percentage contribution of each transporter to the efflux of mitoxantrone was calculated by subtracting the transport activity observed in double transfectants in the absence of any inhibitor from the activity observed in the presence of 5 μM FTC (a specific ABCG2 inhibitor) or 20 μM VBL (a competitive inhibitor of Pgp) alone. The inhibition of efflux activity in the presence of both FTC and VBL was taken as 100%. Double arrows represent mitoxantrone efflux activity contributed by ABCG2 (difference between mitoxantrone alone and in the presence of 5 μM FTC) or Pgp (difference between mitoxantrone alone and in the presence of 20 μM VBL). Both FTC and vinblastine were added together to obtain complete inhibition of efflux (taken as 100% accumulation). Shown here is a graph from a single experiment that was done independently at least three times. Mito, mitoxantrone.