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. 2020 Jan 14;10:218. doi: 10.1038/s41598-019-57076-5

Figure 1.

Figure 1

ALK regulates ETV5 expression through the MAPK signalling pathway. (a) Relative ETV5 mRNA expression levels in four different neuroblastoma cell lines treated for indicated time periods with a vehicle control (DMSO) or the ALK inhibitor, TAE-684 (0.3 µM). (nCLB-GA, SK-N-AS, SH-SY5Y = 4; nNB-1 = 5; mean with error bars representing 95% CI after error propagation with mean centring and scaling to control). (b) Western blot analysis for p-ALK, total ALK, ETV5, p-ERK1/2 and total ERK1/2 in four different neuroblastoma cell lines after ALK inhibition with TAE-684 (0.3 µM, 6 h). (cropped images, full-length images are presented in Supplementary Figs. S5–6). (c) Western blot analysis for p-ALK, total ALK, ETV5 and p-ERK1/2 in IMR-32 after stimulation with ALK ligand (ALKAL1) for 30 min or 6 h and subsequent treatment with ALK inhibitor, crizotinib (0.25 µM). (cropped images, full-length images are presented in Supplementary Fig. S7a). (d) Relative ETV5 mRNA expression levels in non-neuroblastoma ALKoma tumour cell lines Karpas-299 (ALCL) and H3122 (NSCLC) after a 6 h treatment with a vehicle control (DMSO) or the ALK inhibitors, crizotinib (0.5 µM) or TAE-684 (0.3 µM). (n = 1; mean with error bars representing SD after error propagation). (e) Relative Etv5 mRNA expression levels in ALK-dependent Ba/F3 cells 5 h post-treatment with a vehicle control (DMSO) or the ALK inhibitor, TAE-684 (0.3 µM). (n = 1; mean with error bars representing SD after error propagation). (f,g) Relative ETV5 mRNA expression levels in four different neuroblastoma cell lines after a 6 h treatment with a vehicle control (DMSO) or the MEK inhibitor, U-0126 (8 µM) and the PI3K inhibitor, pictilisib (500 nM). (nCLB-GA, NB-1, SH-SY5Y; U-0126 = 5; nSK-N-AS; U-0126 = 4; nCLB-GA, SK-N-AS, SH-SY5Y; pictilisib = 4; nNB-1, pictilisib = 6; mean with error bars representing 95% CI after error propagation with mean centring and scaling to control). (h) Boxplot representation of the log2 ETV5 mRNA expression levels in a large independent primary neuroblastoma cohort (GSE49711 dataset) with (left, 33 cases) and without RAS/MAPK pathway mutations (right, 226 cases) (GSE120572, left panel); and for cases with mutant ALK (ALKmut) or mutations in other RAS/MAPK pathway genes (Mutwithout ALK) (right panel). (*p < 0.05; **p < 0.01; ***p < 0.001).