In cell developer

2 million NCI-H1373 cells were seeded in 10 cm dishes and transduced with pBOB-EF1-FastFUCCI-Puro lentivirus in the presence of 8 μg/mL polybrene. After 24 hours, the media was changed and after 48 hours fresh medium containing 1μg/ml puromycin was added to select infected cells. Cells were selected for 72 hours and then expanded. Red fluorescent cells (i.e. cells in G1) were enriched by flow cytometry using a FACSAria 3 (BD). FastFUCCI expressing cells were seeded into 96-well plates and treated with the indicated concentrations of RBN-2397 and/or tapinarof or CH-223191. After 48 hours, fluorescent cells were scanned using an IN Cell Analyzer 6500 System and then analyzed by IN Cell Developer (Cytiva).

2 million nuclight labeled NCI-H1373 cells were seeded in 10 cm dishes and transduced with pLenti-C-mGFP-AHR lentivirus in the presence of 8 μg/mL polybrene. After 24 hours, the media was changed, and cells were grown for an additional 48 hours before GFP and Nuclight Red dual fluorescent cells were enriched by flow cytometry (BD FACSAria 3). Dually fluorescent cells were seeded into 96-well plates and treated with the indicated concentrations of RBN-2397, Olaparib, tapinarof and/or CH-223191 the next day. After 2 hours of exposure to drugs, fluorescent cells were scanned by IN Cell Analyzer 6500 System and then analyzed by IN Cell Developer (Cytiva). To assess endogenous AHR localization, NCI-H1373 cells were seeded in 96-well plate (5000 cells/well) and treated the next day with the indicated concentrations of RBN-2397, olaparib and/or tapinarof. After 2 hours of exposure to drugs, treated cells were fixed in pre-cooled methanol at −20°C for 20 min, blocked in 3% bovine serum albumin for 15 min, incubated with Anti-AHR (CST, 83200S) antibodies for 1 h, and then incubated with Goat anti-Rabbit IgG Secondary Antibody, Alexa Fluor 488 (ThermoFisher, A-11008) secondary antibodies for 30 min. Final staining with DAPI for 10 minutes. Fluorescent cells were scanned by IN Cell Analyzer 6500 System and then analyzed by IN Cart (Cytiva).

Two orthogonal approaches were used to analyze the cell cycle: EdU incorporation: Two hours prior to harvesting, the cells were treated with 10 μM EdU. After harvesting, EdU incorporation levels were quantified as per manufacturer’s protocol using the Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen C10420) or the Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen C10634). The Attune NxT Flow Cytometer (Thermo Fisher) was used to determine the percentage of EdU-positive S-phase cells in the population. Quantitative FastFUCCI assay: 250,000 AGS, HGC27 or YCC6 cells were seeded in a 6 well dish and transduced with pBOB-EF1-FastFUCCI-Puro lentivirus (RRID:Addgene_86849)(link) in the presence of 8 μg/mL polybrene. After 24 hours, the media was changed and after 48 hours fresh medium containing 2 μg/ml puromycin was added to select infected cells. Cells were selected for 72 hours and then expanded. Red fluorescent cells (i.e. cells in G1) were enriched by flow cytometry using a FACSAria 3 (BD). FastFUCCI expressing cells were seeded into 96 well plates and treated with the indicated concentrations of drug. After 24 hours, fluorescent cells were scanned using an IN Cell Analyzer 6500 System (Cytiva) and analyzed by IN Cell Developer (Cytiva).