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. 2023 Mar 6;42(16):1294–1307. doi: 10.1038/s41388-023-02632-7

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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. 2 — A Common up- or down-regulated genes in CRC tissues and OC-L2HG-stimulated HT29 and HCT116 cells. HT29 and HCT116 cells were treated with 500 μM OC-L2HG for 24 h. B Interactome analysis of transcription factors among the 26 genes upregulated by OC-L2HG. C Analysis of the mRNA expression of ATF4 and ATF4 target genes in HT29 cells treated with OC-L2HG for 24 h, using real-time PCR. Heat map showing the gene expression levels of cells stimulated with the indicated concentrations of OC-L2HG as a scaling ratio relative to unstimulated cells. D Immunofluorescence staining and quantification of ATF4 in HT29 cells treated with 500 μM OC-L2HG. Data represent the mean ± SD of three independent experiments. Scale bar, 200 μm. E ATF4 protein expression in the whole and nuclear fractions of HT29 and HCT116 cells treated with 500 μM OC-L2HG. F Transcriptional activity of ATF4 in HT29 and HCT116 cells treated with 500 μM OC-L2HG (see Methods). Data represent the mean ± SD of three independent experiments. G Effect of ATF4 knockdown on the mRNA expression of ATF4 target genes in HT29 cells treated with 500 μM OC-L2HG. mRNA levels in each sample were converted to a log2 fold-change relative to the control. Red and blue indicate higher and lower levels, respectively, compared to the control (white). H Effect of ATF4 knockdown on the viability of CaR1 cells treated with L2HG (250 μM) under serum-free conditions for 6 d. Viable cells were counted using a trypan blue exclusion assay. Data represent the mean ± SD of three independent experiments. I, J Expression levels of nuclear ATF4 in HT29 cells treated with 500 μM OC-L2HG or 500 μM OC-D2HG for 24 h. Nuclear ATF4 expression was measured using western blotting (I) and immunofluorescence staining (J). Data represent the mean ± SD of three independent experiments. K Transcriptional activity of ATF4 in HT29 and HCT116 cells treated with OC-L2HG or OC-D2HG. Data represent the mean ± SD of three independent experiments.