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. 2023 Oct 20;14:6523. doi: 10.1038/s41467-023-42025-8

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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 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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Fig. 6 — a Identification of METTL16-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS). b, c Western blot verification of the interaction between METTL16 and SIRT2 in HGC-27 cells. b Endogenous METTL16 in HGC-27 cells was precipitated using the anti-METTL16 antibody or IgG (mock IP). Co-precipitated SIRT2 was detected by western blot. c Recombinant His-tagged METTL16 was incubated with GST-SIRT2 or GST proteins at 4^oC for 4 h followed by His pull-down and western blot. d, e SIRT2 inhibited copper-induced METTL16-K229 lactylation. Cell lysates were immunoprecipitated using Flag-M2 beads and western blot for total or lactylation of METTL16 using anti-Flag or METTL16^K229-lac antibody. d Flag-METTL16 and HA-SIRT2 plasmids were co-transfected into HGC27 cells followed by immunoprecipitation. e The Flag-METTL16 plasmid was transfected in control or SIRT2-knockdown (SIRT2-Sh) HGC-27 cells with or without AGK2 (10 μM) treatment for 24 h. f HGC27 cells co-transfected with Flag-METTL16 and HA-SIRT2 were treated with CuCl₂ (5 μM) for 12 h and followed by immunoprecipitation. g The flag-METTL16 plasmid was transfected in control or SIRT2-knockdown (SIRT2-Sh) HGC-27 cells with or without the treatment of AGK2 (10 μM, 24 h) or CuCl₂ (5 μM, 12 h). h SIRT2 was transfected in METTL16-WT/K229R/K229E rescued cells and the relative m⁶A level of FDX1 was detected by MeRIP-qPCR assay (n = 3). P = 0.00371 (WT), 0.3224 (K229R), and 0.8714 (K229E). i SIRT2 was transfected in METTL16-WT/K229R/K229E rescued cells and FDX1 mRNA level was detected by the qRT-PCR assay (n = 3). P = 0.00926 (WT), 0.4222 (K229R), and 0.5107 (K229E). j Different doses of SIRT2 overexpression plasmid were transfected in HGC-27 cells, and the protein level of FDX1 was analyzed by western blot using FDX1 antibody. k Western blot of FDX1 protein level in Ctrl or SIRT2-Sh HGC-27 cells with or without AGK2 treatment. l Immunofluorescent staining of tissue microarray (T16-425) containing 54 pairs of GC and adjacent normal tissues. Representative images showing the co-expression of SIRT2 and FDX1. Pearson correlation analysis of the average optical density (AOD) value between SIRT2 and FDX1 was measured by Image J (R = −0.4536). P = 0.0062. Scale bar, 50 μm. Statistical data presented in this figure show mean values ± SD of three times of independent experiments. Statistical significance was determined by Two-tailed t test, *P < 0.05, **P < 0.01, NS not significant. Source data are provided as a Source data file.