Doxycycline

Flp-In T-Rex HeLa cells containing Aurora B-EGFP constructs were generated following the ThermoFisher protocol (Flp-In™ T-REx™ Core Kit, ThermoFisher K650001; ThermoFisher Scientific). Briefly, cells were co-transfected with Aurora B-GFP constructs (pcDNA5/FRT/TO) and pOG44 (expressing Flp recombinase) in 1:9 ratio using Fugene HD (Promega). Forty-eight hours after transfection, Flp-In T-Rex HeLa cells were put under selection for two weeks in DMEM with 10% tetracycline-free FBS supplemented with 250 μg/mL hygromycin (Sigma-Aldrich) and 5 μg/mL blasticidin (Sigma-Aldrich). The resulting foci were pooled and tested for expression. Gene expression was induced with 1 μg/mL doxycycline (Santa Cruz Biotechnology Europe, Heidelberg, Germany).

Activation of the XBP1-TagRFP biosensor was performed by adding 2 μg/mL doxycycline (Santa Cruz Biotechnology, Dallas, TX, USA) to the medium for two days. ER stress was induced by adding 5–10 μg/mL tunicamycin (Abcam, Cambridge, UK) to the medium for one day. Images were taken using a Nikon Eclipse Ti-E microscope (Nikon, Tokyo, Japan) and NIS Elements software Advanced Research version 4.30.

Luc14 and Gal4-EED/luc HEK 293 cells were grown in Gibco DMEM high glucose 1× (Life Technologies) with 10% Tet-free Fetal Bovine Serum (FBS) (Omega Scientific), 1% penicillin streptomycin (ATCC) at 37 °C in a humidified 5% CO₂ incubator. Gal4-EED/luc cells were treated with 1 µg/mL doxycycline (Santa Cruz Biotechnology) for two days to induce stable polycomb repression. Dox was removed and cells were cultured for another four days before being seeded in 12-well plates. Luc14 cells and dox-induced Gal4-EED/luc cells were seeded in 12-well plates such that cells reached 90% confluency for lipid-mediated transfection. Transfections were performed with 1 µg plasmid per well, 3 µL Lipofectamine LTX, and 1 µL Plus Reagent (Life Technologies) per the manufacturer’s protocol. Seventy-two hours post transfection, cells were either collected for analysis or passaged further.
puromycin selection was carried out on Gal4-AAP-expressing cells for the experiments represented in Figure 5 and Supplemental Figure S1. Dox-treated Gal4-EED/luc cells were transfected in 12-well plates and then grown for 24 h before the addition of 10 µg/mL puromycin (Santa Cruz Biotechnology) to Gibco DMEM high glucose 1× (Life Technologies) with 10% Tet-free Fetal Bovine Serum (FBS) (Omega Scientific), 1% penicillin streptomycin (ATCC). Cells were grown in puromycin containing media for two days before wash out.

Upon infection of EAC and mouse YTN cells with pLKO.1-TRC viral particles, cells were selected using puromycin (Santa Cruz Biotechnology, #205821). ESO26, FLO1, and SKGT4 cells required 3 μg/mL, 4 μg/mL, and 5 μg/mL of puromycin, respectively. YTN5 and YTN16 cells required 8 μg/mL of puromycin. Stable knockdown cells remained in media containing puromycin throughout the length of culture, and knockdown was confirmed using quantitative PCR prior to experiments. CRISPR/Cas9 viral particles were exposed to SKGT4 cells. mCherry+ and GFP+ cells were sorted, and single cells were seeded into 96-well plates. Single colony cells were maintained in culture media with tetracycline-free FBS (Omega Scientific, #FB-15). FOXM1 mutation was induced with the addition of 2.5 μg/mL doxycycline (Santa Cruz Biotechnology, #100929-47-3) for 1 week.

A range of PD-L1 expression cell lines were created and embedded into Histogel as described previously (20 (link), 21 (link)). Briefly, these cells were seeded at 5 x 10⁶ cells per T75 (25 ml of media). The next day cells were treated with increasing amounts of Doxycycline (Santa Cruz Biotechnology, Dallas, Texas, USA) for 24 hours to generate cells with increasing amounts of PD-L1. K562 cells are suspension cells, therefore cells expressing different levels of PD-L1 can be harvested at the same time by centrifugation. Ten percent of the cells were lysed for Western blotting and 90% were subjected Histogel embedding. Cells were washed, resuspended, and fixed in 10% formalin (Thermo Fisher Scientific, Burlington, ON, Canada) by incubating on ice for 60 minutes. During this time, cells were counted using the Moxi Z cell counter. Fixed cells were washed and dried cell pellets were resuspended in 65°C molten Histogel (Thermo Fisher, Canada) at approximately 2x10⁷ cells/100µL of Histogel. The gel-embedded cells were solidified at 4°C and overlaid with 70% ethanol until processing into paraffin-embedded blocks. FFPE cell blocks were then constructed into TMAs. These cell lines not only serve as on-slide controls but the PD-L1 expression for each patient is normalized to the cell line TMA to correct for slight differences in staining across multiple patient TMAs.