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. 2024 Mar 13;10:29. doi: 10.1038/s41421-024-00655-2

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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graphic file with name 41421_2024_655_Figf_HTML.jpg — Figure 3 Calcium-binding protein 39-like (CAB39L) and Sestrin-1 (SESN1) mediated the anticancer effects of metformin through AMP-activated protein kinase–mammalian target of rapamycin (AMPK-mTOR) signaling. (a, b) Altering the levels of CAB39L and SESN1 mimicked the effect of metformin on the AMPK/mTOR signaling axis. (a) Upper panel. Representative western blotting of whole-cell lysates from SUM159PT treated with vehicle or metformin or transfected with the expression vectors coding for CAB39L or for two shRNAs against CAB39L, stained with the indicated pan- and phospho-specific antibodies. Lower panel. Quantitative densitometry of phospho-thr¹⁷² AMPKα calculated from the analysis of three western blottings including that in panel (a). (b) Upper panel. Representative western blotting of similarly stained whole-cell lysates from of the same cells treated as from panel (a) and transfected with the expression vectors for SESN1 or with a siRNA against SESN1. Lower panel. Quantitative densitometry of phospho-thr¹⁷² AMPKα calculated from the analysis of three western blottings including that in panel (b). (c) The LKB1–CAB39L complex was modulated by metformin. Representative western blotting of whole-cell lysates of vehicle- and metformin-treated SUM159PT cells, immunoprecipitated with anti-LKB1 and anti-CAB39L antibodies (upper left and lower left panels, respectively). Actin staining was used as a loading control for the input material (right panel). (d) Metformin stimulates LKB1 kinase activity. Immuno-kinase assay. Histograms showing the enzymatic activity of LKB1: immunoprecipitates from HEK-293 cells treated or not with 0.5 mM of metformin for 24 h. Mean of two independent experiments. Statistics (t-test): P < 0.001.