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. 2022 May 25;13(5):496. doi: 10.1038/s41419-022-04941-3

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© The Author(s) 2022

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. 2 — A KYSE150 cells were transfected with luciferase reporter genes of 16 tumor-associated signaling pathways and renilla luciferase for 24 h. Cells were incubated with DMSO or U0126 (5 μM) for 6 h and luciferase activity was measured using Dual-Luciferase Assay. Log2 FC was assessed by dividing the luciferase signal obtained from U0126 treated cells by that obtained from DMSO-treated ones. Error bars represent the mean ± SD. B KYSE30 and KYSE150 were transfected with SIE and renilla luciferase reporter for 24 h. Cells were incubated with DMSO or U0126 (5 μM) for 6 h and luciferase activity was measured. C Western blotting showed the expression of activated and total signal transducer and activator of transcription 3 (STAT3) and ERK expression in KYSE30 and KYSE150 treated with U0126 in a time-dependent manner. D STAT3 and DAPI staining in KYSE30 and KYSE150 after U0126 (5 μM) treatment for 2 h. The fluorescence signal intensity of STAT3 and DAPI was analyzed using the ZEN2.3 software. Scale bar, 50 μm. E Western blotting showed the expression of pSTAT3 and pERK in KYSE450, KYSE510, TE1 and TE3 treated with U0126 in a time-dependent manner. F Western blotting shows the expression of pSTAT3 in wild-type (WT), ERK1-KO, ERK2-KO, and ERK1/2-DKO ESCC cells. G Western blotting shows the expression of pERK in WT and STAT3-knockout (KO) cells. Densitometry analyses of pSTAT3 or pERK expression were performed. Error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. H Western blotting showed the phosphorylated and total STAT3 and ERK expression in 12 human ESCC tissue. Densitometry analyses and Pearson correlation showed a negative correlation for pSTAT3 and pERK expression. I Kaplan–Meier analysis was performed to evaluate the effects of pSTAT3 expression on overall survival (n = 301). J Kaplan–Meier analysis was performed to evaluate the effects of pERK associated with pSTAT3 expression on overall survival (n = 290). Group +/+: dual high expression level of pERK and pSTAT3; group +/−: high expression of pERK and low expression of pSTAT3 or low expression of pERK and high expression of pSTAT3; group −/−: dual low expression of pERK and pSTAT3.