Fig. 4: Lactate shuttling-mediated NPM1 lactylation inhibits SLC7A11 expression and induces ferroptosis during kidney IRI. | Nature Communications

Fig. 4: Lactate shuttling-mediated NPM1 lactylation inhibits SLC7A11 expression and induces ferroptosis during kidney IRI.

From: Nucleophosmin 1 lactylation in graft kidney induces ferroptotic trigger waves that exacerbate delayed graft function

Fig. 4

a, b Global lactylation of HK-2 cells cocultured with different CM or lactate for 24 h; IR-CM-12h represents the CM of HK-2 cells experienced ischemia and reperfusion for 12 h (n = 3 group−1). c Bubble plot of significantly enriched GO terms (Biological Process, BP); Fisher’s exact test. d The top 30 proteins with the highest lactylation intensity from the lactylome analysis and the functional enrichment analysis for metabolism and transcriptional regulation. e Venn diagram of lactylated proteins enriched in cystine/cysteine/GSH metabolic pathway and metabolism & transcriptional regulation pathway. IP assays determining endogenous NPM1 lactylation in mouse kidney tissues (f, n = 6 group−1; g, n = 20 group−1) and HK-2 cells stimulated with different CM (h, i, n = 3 group−1). j NPM1 lactylation in endogenous NPM1-knockout (NKO) HK-2 cells reconstituted with full-length NPM1 or its site-directed mutants (n = 3 group−1). k MS analysis identified residue lysine 257 (K257) as the key lactylation site. l KEGG analysis displaying the top 20 enriched pathways of differentially expressed genes (DEGs) from RNA sequencing in NKO-NPM1 WT versus NKO-NPM1 K257R cells (n = 6 versus 6). m Volcano plot highlighting DEGs enriched in ferroptosis pathway (n = 12). n, o Translational and transcriptional levels of ferroptosis-related genes in NKO HK-2 cells expressing either WT or K257R NPM1, following treatment with or without 10 mM sodium lactate (n = 3 group−1); normalized to negative control group results; one-way ANOVA followed by Tukey’s test. p ChIP‒sequencing tracks for SLC7A11 in NKO HK-2 cells reconstituted with WT or K257R NPM1. q ChIP-qPCR was conducted using anti-NPM1 antibody to assess the enrichment of WT or K257R NPM1 at the SLC7A11 promoter in NKO HK-2 cell (n = 3 group−1); one-way ANOVA followed by Tukey’s test. r Lipid peroxidation in HK-2 cells overexpressing WT or K257R NPM (n = 3 group−1). Data are presented as mean ± SD. All experiments were repeated at least three times, yielding similar results. The pvalues are shown for the indicated comparisons. Source data are provided as a Source Data file.

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