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. 2021 Aug 3;40(39):5814–5828. doi: 10.1038/s41388-021-01978-0

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© The Author(s) 2021

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. 7 — A, B Aberrant overexpression of GRB2 RNA in ESCC tumors than in paired normal tissues by qRT-PCR (n = 215; A) and by RNA-Seq (n = 7; B). C, D Kaplan–Meier estimates of survival time of patients in SYSUCC cohort (n = 215) by different GRB2 RNA levels (C) or m⁵C levels (D) in tumors. Median survival time for patients with high GRB2 RNA or m⁵C levels (≥median) was 33.6 months or 33.6 months compared with 85.9 or 88.4 months in patients with low GRB2 RNA (C) or m⁵C levels (D) (<median), with the adjusted HRs of 1.78 (95% CI, 1.22–2.61) and 1.70 (95% CI, 1.16–2.49), respectively. E Western blotting assays showing higher protein levels of GRB2 in ESCC tumors than in paired normal tissues (n = 10). F Representative immunohistochemical staining (IHC) images of GRB2 protein in ESCC tumors and in paired normal tissues. Scale bar, 500 μm (left panel) and 100 μm (right panel). G GRB2 protein levels were higher in ESCC tumors than in paired normal tissues (n = 59) as indicated by the IHC score. H Kaplan–Meier estimates of survival time of patients by different GRB2 protein levels in tumors. The median survival time for patients with high GRB2 (IHC score > 6) was 22.8 months compared with 85.4 months in patients with low GRB2 (IHC score ≤ 6), with the adjusted HRs of 3.64 (95% CI, 1.57–8.41). I Representative images showing positive correlations between protein levels of NSUN2 and GRB2 in ESCC specimens. Scale bar, 100 μm. J Statistical analysis of IHC staining showing the percentage of ESCC specimens displaying higher or lower NSUN2 levels and the corresponding GRB2 expression. K Spearman’s correlation analysis between protein levels of NSUN2 and GRB2 in ESCC tumors (n = 59). L A proposed model for the regulatory mechanism of E2F1-NSUN2-m⁵C/LIN28B-GRB2-PI3K/AKT and ERK/MAPK signaling axes in the tumorigenesis and progression of ESCC. Data represent as mean ± SEM in (A), boxplots in (B) and violin plots in (G). The centerlines of the boxplots and violin plots represent median, while the upper and lower hinges indicate 25th and 75th percentiles, respectively. P values were calculated using the two-sided paired Wilcoxon signed-rank test in (A) and (G), two-sided paired t-test in (B), two-sided Chi-square test in (J) and two-sided log-rank test in (C), (D) and (H). ACTIN served as a control in (E).