The cell line LU-HNSCC-26 (HN26) was previously isolated from a low-grade, oropharyngeal, p16 positive, squamous cell tonsillar stage II (T2N0M0 ) carcinoma . The cells were thawed from single tandem repeat verified batches  and used within 18 passages. They were cultured in R10 medium (RPMI 1640 with stable glutamine supplemented with 1 mmol/L sodium pyruvate, 1×MEM non-essential amino acids, 20 µg/mL gentamicin and 10% fetal bovine serum (FBS), all from GE Healthcare (Piscataway, NJ, USA)) in a humidified 37°C incubator with 5% CO2.
Bortezomib, carfilzomib, ixazomib and RG7112 were purchased from Selleck Chemicals (Houston, USA) and cisplatin from Sandoz AS (Copenhagen, Denmark).
Determination of drug sensitivity
Dose response curves were generated for each substance individually. Cells were seeded in 96-well plates (6,000 cells/well). After incubation for 48 h, they were treated with increasing concentrations of the drugs as indicated in the figures. After 5 days of incubation, the number of cells were measured by the sulforhodamine B (SRB) assay as previously described . Dose response curves were generated by fitting the data to sigmoidal dose-response curves and half maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism version 7 (GraphPad Software, La Jolla, CA).
For each experiment, cells were seeded in ten 96-well plates (6,000 cells/well). Each plate was first treated with one of the concentrations of cisplatin (0 and a serial dilution ranging from 0.05 to 50 µmol/L) for 1 h, and, after siphoning off the medium with cisplatin, with a serial dilution of the respective combination drug (bortezomib: 0.84 to 59.5 nmol/L, carfilzomib: 0.20 to 200 nmol/L, ixazomib: 0.40 to 400 nmol/L, RG7112: 0.010 to 100 µmol/L). Each combination of concentrations was repeated in 6 wells. The plates were incubated with the inhibitor for 5 days followed by the SRB assay to determine cell numbers. The STR result were fitted to sigmoidal dose-response curves, and IC50 values with 95% confidence intervals were calculated using GraphPad Prism. These parameters were calculated for both substances in each combination pair, determining the IC50 values for each substance in combination with each concentration of the other. Only data that fitted the sigmoidal equation with an R2 coefficient higher than 0.95 were used in the final analysis.
Western blot analysis
HN26 cells were treated as indicated after which attached cells were washed once in cold PBS and lysed in radioimmunoprecipitation assay buffer: 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mmol/L NaCl, 2 mmol/L Na3VO4, 1 mmol/L NaF, 20 mmol/L Na4P2O7, complete protease inhibitors with EDTA (Roche Applied Science, Basel, Switzerland), and 50 mmol/L Tris-HCl, pH 7.4. The protein concentration of the lysates was determined by the Micro Bicinchoninic Acid protein assay (Thermo Scientific, Rockford, IL). Equivalent quantities of protein were electrophoresed on 4–12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to polyvinylidene fluoride membranes. Specific proteins were detected with the indicated antibodies and the ECL prime chemiluminescence detection system (GE Healthcare, Fairfield, CT). The antibodies used were anti-p53 (#2527), anti-p21 (#2947), anti- caspase-3 (#9665), and anti-ERCC-1 (#5437) from Cell Signaling Technology (Danvers, MA) and PARP1 (#sc-7150) Santa Cruz Biotechnology (Santa Cruz, CA). To control for gel-loading, the membranes were stained with 0.1% Coomassie R-350 in 50% methanol followed by quantification of the total protein content in each lane by densitometry . A C33A2 cell lysate was used as positive control for p53. The C33A2 cell line is derived from the HPV-negative cervical cancer cell line C33A and has been described previously .
Cell cycle analysis
Cells were seeded in cell culture flasks and treated with the different proteasome inhibitors and the MDM2-inhibitor in combination with cisplatin and then fixed at different time points as indicated. After treatment, attached cells were trypsinized and mixed with floating cells, collected by centrifugation, followed by fixation with ice cold 70% ethanol. Cells were then stained with 50 µg/mL propidium iodide and analyzed on a FACS Calibur flow cytometer (Becton, Dickinson, San Jose, CA, USA) connected to a computer running CellQuest (Becton Dickinson) data collection software. The respective cell cycle phases were evaluated using ModFit LT software, version 3.1 (Verity Software House, Topsham, ME, USA).