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. 2020 Sep 25;39(44):6856–6870. doi: 10.1038/s41388-020-01465-y

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Combination Index (CI) was calculated in order to assess the combined effects of drugs (CompuSyn software, v.1.0). a–c Graphs illustrating CI results for fractions affected by combined application of indicated drugs (ERLO: erlotinib; GEFI: gefitinib; CP: cisplatin; GEM: gemcitabine) on SCaBER cells after 72 h. Drugs were used at concentrations of 4x IC₅₀, 2× IC₅₀, 1× IC₅₀, 0.5× IC_50, 0.25× IC₅₀, 0.125× IC₅₀. CI = 1: additive effect, CI < 1: synergism, CI > 1: antagonism. d Fractions affected of FaDu cells by combined application of indicated drugs (ERLO: erlotinib; CP: cisplatin; GEM: gemcitabine). Drugs were used at concentrations of 4× IC₅₀, 2× IC₅₀, 1× IC₅₀, 0.5× IC_50, 0.25× IC₅₀, 0.125× IC₅₀. CI = 1: additive effect, CI < 1: synergism, CI > 1: antagonism. Data represent means from at least n = 3 independent experiments. e Polygonograms summarizing the effects (synergism/antagonism) according to Chou 2010 [32] of drug combinations for erlotinib, gefinitinib, cisplatin, and gemcitabine on SCaBER and FaDu cells. FaDu served as a technical “squamous” control cell line. f Applied drugs and concentrations for EGFR signaling studies. g Representative western blot analyses illustrate activation and inhibition of EGFR/p-EGFR (Tyr1068), ERK / p-ERK (Thr202, Tyr204), and p-AKT (Ser473) 24 h after treatment with indicated drug concentrations and combinations. DMSO application was used as an untreated control. β-actin served as loading control. h–l Relative mRNA expression of the EGFR target gene SOX9, the ERBB receptor ERBB4, and ERBB ligands (AREG, EREG, and HB-EGF) normalized to corresponding DMSO control is shown 24 h after treatment of SCaBER cells with indicated drugs. GAPDH was used for standardization. FC: fold change. Vertical lines: +SEM of triplicates. Data (western blot and mRNA expression) were confirmed by n = 3 independent experiments.