96 well opaque bottom plates

Cytotoxicity was measured utilizing the cell viability luminescence assay (CellTiter Glo; Promega; Madison, WI). Cells were plated in 96-well opaque bottom plates (BD-Falcon) at a density of 1,000 cells/well and were allowed to attach overnight before being exposed to the drugs of interest. Stock solutions of cytotoxic drugs SN-38, paclitaxel, and doxorubicin, known to be substrates for ABCG2, P-gp, and MRP1, respectively, with and without their respective inhibitors, were prepared in cell-type specific media. Cells were incubated in serial dilutions of the drugs for 72 h at which point cell growth was assayed as described previously. Luminescence was measured on a Tecan Infinite 200 Pro (Mannedorf, Switzerland) plate reader. Cytotoxicity (IC₅₀) was defined as the drug concentration at which the cell viability was reduced to 50% of the untreated control, and was calculated as the mean of three separate observations.

The BacMam-fLuc virus was added to 2.5 × 10⁶ cells at a titer of 1:50 viral particles per cell, unless otherwise specified, in 3 mL of media and incubated for 1hr at 37 °C. 20 mL of media were subsequently added, and cells were plated into 96-well opaque bottom plates (BD-Falcon) at a density of 10,000 cells/well and incubated for 24 h in a 37 °C incubator. Media were then aspirated and 100 μL luciferin, with and without Ko143, dissolved in IMDM media with 5% FBS was added. Plates were read on a Tecan Infinite 200 Pro plate reader.