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. 2023 Nov 29;14:7863. doi: 10.1038/s41467-023-43670-9

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© The Author(s) 2023

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 Consensus binding motif of ELF3. b, c Pearson correlation analysis of Spink1/SPINK1 and Elf3/ELF3 mRNA expression in b NRasV12+Myr-AKT HCC mouse model in a stepwise manner from normal to early and advanced HCC indicated by ●, ■, ▲, ▼, ○ and □, and c human samples from TCGA-LIHC dataset. d Co-staining of SPINK1 (yellow) and ELF3 (green) protein in human HCC tumor with high and low SPINK1 expression by multiplex IHC. DAPI (blue), nucleus. Scale bar: 50 μm. e (Top) Computational prediction of ELF3 binding sites (S1 at -4053 to -3802 bp and S2 at -2103 to -2019 bp) on SPINK1 promoter region by Gene Transcription Regulation Database (GTRD)³³. TSS = transcription start site. (Bottom) Reporter constructs were generated by cloning of full-length (FL) or truncated promoter fragments (pT1 to pT3) into a pGL3 vector encoding firefly luciferase. f Luciferase reporter assays showing relative luciferase unit (firefly/renilla) in Huh7 cells with FL or different truncations of SPINK1 (pT1 to pT3) promoters. pRL-CMV Renilla luciferase plasmid co-transfected for normalization. g ChIP-qPCR confirmation of ELF3 binding to predicted sites (S1 and S2) on SPINK1 promoter in Huh7 cells, by using ELF3 and IgG antibodies. h–j, h Relative luciferase reporter activity, i mRNA expression and j secretory SPINK1 protein levels in Huh7 cells with or without CRISPR/Cas9-mediated ELF3 knockout (shELF3-2 and shELF3-3). k Relative luciferase reporter activity, l mRNA expression, and m secretory SPINK1 protein levels in MHCC97L cells with or without shELF3-2 and shELF3-3, and treated with 5-FU, cisplatin or DMSO. b n = 2-3 mice; c n = 371; d n = 2 human HCC samples with high SPINK1, 2 human HCC samples with low SPINK1; f–j n = 3 independent experiments; k, l n = 4 independent experiments; m n = 3 independent experiments. Data were expressed as mean ± s.e.m. Statistical analysis: b, c two-sided Pearson correlation analysis, (f, h–j) one-way ANOVA, or (g, k–m) two-way ANOVA. Source data are provided as a Source Data file.