FIGURE 7.

Short term treatment with P-gp substrates or modulators is sufficient to rescue the mutant protein to the cell surface. A, time course of recovery of cell surface expression of D164C/D805C mutant P-gp upon short term treatment with CsA is plotted. The transduced cells were grown for 24 h after which the medium was replaced by fresh DMEM with 15 μm CsA. The cell surface expression of D164C/D805C was monitored over regular time intervals for 8 h. The expression of double (D164C/D805C) mutant at the cell surface after 18 h treatment with CsA during growth was taken as 100%. The values represent the average of two independent experiments. B, recovery of cell surface expression of D164C/D805C mutant-Pgp by CsA in the presence of CYH. Transduced HeLa cells were grown for 24 h, and then the medium was replaced by DMEM with CYH (5 μm), a de novo protein synthesis inhibitor. After 2 h the rescue was initiated by the addition of 15 μm CsA, and the cells were allowed to grow for another 4 h. The recovery by 15 μm CsA at 4 h in the absence of CYH was taken as 100%. The values are plotted as an average from two independent experiments. C, rescue of D164C/D805C to the cell surface by incubation with 15–25 μm of several substrates and modulators of P-gp was measured at 4 h. The values are the average of two independent experiments. XR, tariquidar; VBL, vinblastine; Nilo, nilotinib; Val, valinomycin; Thaps, thapsigargin. D, rescue of D164C/D805C when treated with lower concentrations (25, 250, 350 nm) of either CsA (filled bars) or FK506 (empty bars) for 4 h. The level of rescue in the presence of 15 μm CsA or FK506, respectively, for 4 h was taken as 100%. Data points are plotted as the mean ± S.E. (n = 4).