In vitro cytotoxicity and proliferation assays

Cells were plated at 1000 cells/well in 96 half-well plates. Serial dilutions of drugs or anti-LGR5 ADC were added and allowed to incubate at 37°C for 3–4 days as indicated. For experiments using Rho inhibitor I or tariquidar, cells were pretreated for approximately 24 hours and 1 hour prior to drug or ADC treatment, respectively. Cell cytotoxicity was measured using CellTiter-Glo (Promega) according to manufacturer’s protocol. Luminescence was measured using EnVision mulitlabel plate reader (PerkinElmer). For proliferation studies, measurements were acquired once a day for 4–5 days (n =3–4 experiments). Each condition was tested in at least triplicates. Cytotoxicity data is shown as a single experiment representative of 3–4 independent experiments.