Fig. 3: ABCB1 inhibition by Cyclo-(L-Trp-L-Trp) in human cells.

a Western blot analysis of ABCB1 expression. Proteins were extracted from HEK293T (control), HEK293T ABCB1 (the cells stably expressing exogenous human ABCB1), and HCT-15 cells. Tubulin was used as a loading control. b Flow cytometry analysis of doxorubicin (Doxo) accumulation. HEK293T control or ABCB1 expressing cells were pre-treated with or without 5 µM verapamil (Vera) or 1 mM of Cyclo-(L-Trp-L-Trp), Cyclo-(L-Leu-L-Trp), Cyclo-(L-Trp-L-Pro), or Cyclo-(L-Leu-L-Pro) before incubating with 10 µM Doxo. Bar graphs represent the mean ( ± SD of four independent experiments) fluorescent intensity of Doxo relative to the Vera-treated cells (Vera, the second bar in each cell line). Gray bars: HEK293T cells; white bars: HEK293T ABCB1 expressing cells. c Flow cytometry analysis of doxorubicin (Doxo) accumulation. HCT-15 cells were pre-treated with or without 5 µM Vera or 1 mM of Cyclo-(L-Trp-L-Trp), Cyclo-(L-Leu-L-Trp), Cyclo-(L-Trp-L-Pro), or Cyclo-(L-Leu-L-Pro) before incubating with 10 µM Doxo. Bar graphs represent the mean ( ± SD of three independent experiments) fluorescent intensity of Doxo relative to the Vera-treated cells (Vera, the second bar). d Western blot analysis of DNA damage response. HCT-15 cells were pre-treated with or without 5 µM Vera or 1 mM Cyclo-(L-Trp-L-Trp) before incubating with 0.3 µM Doxo for an additional 3 hours. Tubulin was used as a loading control.