Functional efflux assay

Caco-2 spheroids were serum starved for half an hour in serum free EMEM before conducting the transport experiments. Spheroids were incubated with either 5uM rhodamine 123 (ThermoFisher Scientific, Hampton, NH, USA) or BODIPY FL prazosin (Life Technologies, Carlsbad, CA, USA) or fluorescein methotrexate (Life Technologies, Carlsbad, CA, USA) in serum free EMEM. Inhibition studies were performed by first incubating the spheroids with 15 uM Cyclosporine A (Sigma-Aldrich, St. Louis, MO, USA) or 50 uM Ko 143 (Abcam, Cambridge, UK) or 2 μM fumitremorgin C (Abcam, Cambridge, UK) for 30 minutes at 37 °C. After removing the inhibitor solution the spheroids were then incubated with both substrate and inhibitor for 90 minutes. Spheroids were imaged at different time points using a confocal microscope. Fluorescence intensities were measured in the lumen of spheroids while keeping laser power and exposure time constant. Calibration curves of fluorescence intensities vs. drug concentration were used to calculate concentration inside the lumen. All experiments were conducted by taking the average of n = 10 spheroids per condition.