Spark reader

In order to analyze cell proliferation, cells were seeded in 96-well plates (Sarstedt, Nürnbrecht, Germany) in the following fashion: SCC154 20,000 cells/100 µL/well; Cal27 5000 cells/100 µL/well. A set of five replicates for each dose was used. One day after seeding, cells were treated with 100 µL of inhibitor diluted in DMEM. Ascending inhibitor concentrations were used and ranged between 0.625 and 10 µM. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, D8418, St. Louis, MO, USA) treated cells served as control. Furthermore, cells were irradiated at 2, 4, and 8 Gy (YXLON International GmbH, Hamburg, Germany). The Synergyfinder tool (https://synergyfinder.org, accessed on 31 January 2021) was used for calculating synergistic/additive effects of irradiation and ICG-001 treatment.
After 72 h of treatment, the medium was removed, 100 µL of 56 µM Resazurin (Sigma-Aldrich, St. Louis, MO, USA), diluted in DMEM, was added to each well, and the cells were incubated. The measurements were performed with a TECAN Spark reader (Tecan Group, Männedorf, Switzerland); for Cal27 after 90 min of incubation and for SCC154 after 150 min.
Based on IC₅₀ inhibitor values calculated in the cytotoxicity assay, corresponding inhibitor concentration ranges of ICG-001 were used in subsequent experiments for two cell lines.

The cytotoxic effects of MK were evaluated in a dose–response assay. Cells were seeded into 96-well plates (Sarstedt, Nurnbrecht, Germany). SCC154 (10,000 cells/well), FaDu and Cal27 (5000 cells/well) were seeded in 100 μL DMEM and incubated for 24 h. Following the incubation, cells were exposed to increasing concentrations of MK, previously dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, Missouri, USA). The inhibitor was diluted in cell culture medium in following concentration ranges: 3.75–60 μM for FaDu and Cal27 and 2.5–40 μM for SCC154 cells. For each dose and the vehicle control (0.1% DMSO), five replicates were obtained. Besides MK treatment, cells were irradiated at 2, 4, and 8 Gray (Gy) utilizing the YXLON device (YXLON, International GmbH, Hamburg, Germany). After treatment with MK and radiotherapy, cells were incubated for 72 h. Next, the medium was removed and replaced with 100 μL of a 56 μM resazurin (Sigma-Aldrich, St. Louis, Missouri, USA) solution. FaDu and Cal27 cell lines were incubated for 90 min, while SCC154 were incubated for 3 h. Absorbance measurements were performed using the TECAN Spark reader (TECAN Spark, Tecan Group Ltd, Maennedorf, Switzerland). The half maximal inhibitory concentration (IC50) of MK was calculated and used for subsequent experiments.

In order to determine the cytototoxic effects of PF and calculate its half-maximal inhibitory concentration (IC50) value of PF, we performed a dose–response assay in 96-well plates (Sarstedt, Nurnbrecht, Germany) for each cell line. In particular, 5000 cells/well were seeded in 100 μl DMEM. After 24 h (h) of incubation, cells were exposed to gradually increasing concentrations of the inhibitor. The inhibitor was reconstituted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, Missouri, USA) and diluted in cell culture medium (2.5–40 μM for FaDu and Cal27, and 1.25–20 μM for SCC154 with five replicates per dose). Vehicle control was conducted using 0.1% DMSO. PF treatment was followed by irradiation with 2, 4, and 8 Gray (Gy) with the YXLON device (YXLON, International GmbH, Hamburg, Germany). After 72 h, the medium containing the inhibitor/vehicle control was removed and replaced with 100 μl of 56 μM resazurin (Sigma-Aldrich, St. Louis, Missouri, USA) solution. FaDu and Cal27 were then incubated for 90 min (min), while SCC154 were incubated for 180 min. Measurements were performed using the TECAN Spark reader (TECAN Spark, Tecan Group Ltd, Maennedorf, Switzerland).