Assay to check for cytotoxicity

AQP1-expressing CHO cells were grown overnight on 96-well plates (15–20 000 cells/well). Cells were washed with PBS twice and then incubated with compounds in 100 μL of PBS at room temperature for 30 min. Cytotoxicity effect of compounds on the AQP1-CHO cells was determined using CytoTox-One kit from Promega. CytoTox-One reagent (100 μL) was added to each well and incubated at 22 °C for 10 min. Stop solution (50 μL) was added to each well and mixed by shaking the plate for 10 seconds. Fluorescence was measured using a Fluorocount Microplate Fluorometer (Packard Instrument Company, Meriden, CT, USA.) with an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Maximum LDH release was calculated as the percent cytotoxicity for a given compound by comparing the LDH release to the maximum LDH release by known toxic compound.