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. 2021 Nov 2;12:6314. doi: 10.1038/s41467-021-26718-6

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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. 3 — a Scheme of tRNA m¹A-seq. b Comparison of misincorporation signals caused by m¹A58 in tRNAs between liver CSC and non-CSC cells. Red dots represent tRNAs with elevated m¹A58 methylation levels in CSCs. c Comparison of translation efficiency (TE) between liver CSC and non-CSC cells. Red dots represent genes with increased TE, while black dots represent genes with decreased TE in liver CSCs. d Frequency of codons corresponding to m¹A58 elevated tRNAs was higher in the genes with increased translation efficiency (TE) in CSCs. e Unbiased pathway analysis of TE upregulated genes in liver CSCs compared with non-CSCs. f Western blotting analysis of PPARδ expression in indicated CSCs. n = 4 biologically independent samples. g Effect of elevated m¹A58 methylation levels in tRNA^Ala(AGC) and tRNA^Glu(CTC) on protein synthesis. Reporter vectors were transfected into Mock overexpressed PLC/PRF/5 cells, WT-TRMT6/TRMT61A or Mut-TRMT6/TRMT61A overexpressed PLC/PRF/5 cells, respectively. Control vectors were also transfected into these three cell lines to normalize translation differences among these cell lines. The expression level of two synonymous codon control Ala-(GCG) and Glu-(GAA) remained unchanged upon TRMT6/TRMT61A expression level manipulation. n = 6 biologically independent samples. Exact P values from left to right: 0.00021, 0.27, 0.45, 0.00086, 0.21, 0.15. h Representative immunohistochemistry images of PPARδ (brown) in our cohort of 191 HCC samples. n = 3 independent samples. Scores 0 represents PPARδ staining that was considered negative, Scores 1 represents PPARδ staining that was considered negative, whereas scores 2 and 3 represent PPARδ staining that was considered positive. Grade I (n = 56), Grade II (n = 87), Grade III (n = 48). Scale bar, 100 μm. i Kaplan–Meier plots of overall survival for PPARδ in TMA cohort. Median expression value was used as a cut-off. P values for Kaplan–Meier curves were determined using a two-sided log-rank test. ***P < 0.001, and NS not significant (P > 0.05) by two-tailed Student’s t-test.