DOX efflux assay

Cells (5×10³ per well) were distributed into 96-well plates and incubated for 24 h before being pretreated with 25 μM DOX at 37°C for 2 h, after which time the cells were supplemented with reversal agents (Icory or VRP). After various time points (0, 30, 70, 130 min), the cell culture supernatant was discarded, and the cell cultures were immediately washed three times with ice-cold PBS. The cells were fixed with paraformaldehyde for the detection of radioactivity, in a routine manner.