Fig. 8

EGFR signaling essentially contributes to the protective FGFRi-induced resistance response. A Pathway activity inference derived from the three DEG comparison groups (IR, 6 Gy X-rays; FGFRi, FGFR inhibitor; FGFRi/IR, FGFR inhibitor plus 6 Gy X-rays) per cell model (n = 4) using PROGENy. Rows are clustered hierarchically. B Western blots of phosphorylated EGFR (Y1173) and ERK1/2 (Thr202/Tyr204) in whole cell lysates from 3D lrECM cell models treated as indicated. Vinculin served as loading control. Representative blots are shown. C Densitometric analysis of western blot results shown in ‘B’. Mean fold changes (± standard deviation) compared to corresponding non-irradiated/irradiated control are shown (n = 3). (Two-way ANOVA utilizing normalized densitometry data, Tukey multiple comparison test, ***p ≤ 0.001). D 24-h time kinetic of indicated EGFR phosphoforms in whole cell lysates from treated 3D lrECM UM-SCC 10a cell cultures. Vinculin served as loading control. Representative blots are shown. E Densitometric analysis of western blot data shown in ‘D’. Mean fold changes (± standard deviation) compared to the corresponding control are shown (n = 3). F Cell viability of CRISPR/Cas9-mediated EGFR-knockout cells (ko1, ko2) and corresponding controls after FGFRi treatment under non-irradiated and 6 Gy X-ray irradiated conditions. Bars represent mean cell viability (n = 3; two-way ANOVA; Tukey multiple comparison test; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05). G Cell viabilities of UM-SCC 10a EGFR-knockout cells (ko1) reconstituted with either EGFR wild-type (wt) or kinase-dead (kd) constructs upon FGFRi treatment under non-irradiated and 6 Gy X-ray irradiated conditions. Parental cells and empty-vector-transduced cells were used as controls. Bars represent mean cell viability (n = 2). H Cell viability of UM-SCC 10a cells upon siRNA-mediated knockdown of indicated target genes alone or in combination with FGFRi treatment. Bars represent mean cell viability (n = 3; two-way ANOVA; Tukey multiple comparison test; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05). Non-targeting siRNAs and DMSO were used as controls. I Effectiveness plot of adapter protein RNAi screen shown in ‘G’ (y-axis: cell viability; derived from Fig. 8G; x-axis: EGFR (Y1173) phosphorylation; derived from Fig. S15C, grey bars). Respective ratios to corresponding controls were calculated and -log2 transformed