Figure 2.

H₂O₂ facilitated dynamin-dependent P-gp internalization in hCMEC/D3 cells. The effect of H₂O₂ on P-gp and MRP1 protein expression amounts in whole-cell lysate (a, b) and plasma membrane fraction (c, d) was examined. hCMEC/D3 cells were exposed to 0.25 mM to 5 mM H₂O₂ for 20 min in ECF buffer. Whole-cell lysate and plasma membrane fraction of hCMEC/D3 cells were digested with lysyl endopeptidase and trypsin. The digests were subjected to LC-MS/MS together with internal standard peptides. Each value represents the mean ± SD of 8–16 SRM/MRM transitions in three to four independent samples. One-way ANOVA with Bonferroni-corrected Student's t-test was performed to determine the statistical significance of differences between protein expression levels under control and H₂O₂-treated conditions. (e) Dynasore attenuated the decrease of P-gp protein expression amount in the plasma membrane fraction caused by 0.5 mM H₂O₂ exposure. hCMEC/D3 cells were pre-incubated with dynasore (80 µM) or DMSO (vehicle) for 30 min, and then co-incubated with 0.5 mM H₂O₂ and dyansore or DMSO in ECF buffer for 20 min at 37℃. P-gp protein expression amounts in plasma membrane fraction were determined by LC-MS/MS analysis as described above. Each value represents the mean ± SD of 9-12 SRM/MRM transitions in three independent samples. (f) Dynasore attenuated the decrease of P-gp efflux transport activity caused by 0.5 mM H₂O₂ treatment. hCMEC/D3 cells were pre-incubated with dynasore (80 µM) for 30 min, and then the cellular uptake of vinblastine was measured with or without 0.5 mM H₂O₂, PSC833 and dynasore for 20 min. Each value represents the mean ± SD (n = 3). *p < 0.05, ***p < 0.005 (Student’s t-test).