HNSCC Cell Line Culturing and Inhibitor Treatments

All cell lines were grown in RPMI (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Biochrom AG, Berlin, Germany) at 37°C, 5% CO₂ and 100% humidification. HPV-positive HNSCC cells UD-SCC-2, UM-SCC-47 and UPCI-SCC-154, UPCI-SCC-90, 93VU-147T, UT-SCC-45, and normal human fibroblasts F184 were described previously (21 (link), 33 (link), 39 (link)). Tumor cell lines were identified by a short tandem repeat multiplex assay (Applied Biosystems, Waltham, MD, USA). PARP inhibition was performed using 1 µM olaparib (MyBiosource, San Diego, CA, USA). Wee1 inhibition was performed using 240 nM adavosertib (Selleckchem, Houston, TX, USA) and combined Wee1/Chk1 inhibition was performed at a dose of 60 nM adavosertib and 1 nM prexasertib (MedChemExpress, Monmouth Junction, NJ, USA) unless stated otherwise. Supplementation with nucleosides (EmbryoMax 100×, Sigma-Aldrich, St. Louis, MO, USA) was performed at a final dilution of 1/12.5.