Ver 155008

The HNSCC cell lines Detroit 562 and OECM-1 were maintained at 37 °C in 5% CO₂ in MEM (for Detroit 562) or RPMI 1640 (for OECM-1) (Invitrogen, Carlsbad, CA, USA), supplemented with 10% FBS (Invitrogen), 100 U/mL penicillin and 100 μg/mL streptomycin (both from Invitrogen). HNSCC cells were treated with mock (DMSO only), 40 μM RETRA (a p73 activator, Santa Cruz, CA, USA), 100 μM NSC59984 (a p73 activator, Sigma-Aldrich, St. Louis, MO, USA), 10 μM EX527 (a SIRT1 inhibitor, Sigma-Aldrich, St. Louis, MO, USA), 10 μM VER-155008 (an HSP70 inhibitor, MedChemExpress, Monmouth Junction, NJ, USA) or 10 μM FK886 (an NAMPT inhibitor, MedChemExpress, Monmouth Junction, NJ, USA).

Cells were treated with inhibitor (or solvent control) for one hour at the specified concentrations in the appropriate medium prior to infection (pretreatment). Pretreatment was removed and cells were subsequently infected as described. Fresh medium containing inhibitor (or solvent control) was reapplied. HA15 (MedChemExpress, HY-100437, Monmouth Junction, NJ, USA), VER-155008 (MedChemExpress, HY-10941, Monmouth Junction, NJ, USA), BTB06584 (MedChemExpress, HY-15877, Monmouth Junction, NJ, USA), and Enterostatin (Abbiotec, 350165, Escondido, CA, USA) were dissolved in DMSO to a final concentration of 50mM. Metformin (MedChemExpress, HY-17471A, Monmouth Junction, NJ, USA) and Fluvastatin (MedChemExpress, HY-14664A, Monmouth Junction, NJ, USA) were dissolved in water to a final concentration of 100 mM.