Fig. 4: SLG accumulates after GLO2 downregulation and directly induces nonenzymatic d-lactylation.
a Diagram of GSH-dependent glyoxalase pathway and the corresponding pharmacological inhibitors. b Enzymatic activity detection of GLO2 in innate immune cells stimulated as indicated. c Targeted LC–MS quantification of SLG in BMDMs stimulated as indicated. d Relative quantification of D-Lactate in BMDMs stimulated as indicated. e LC–MS quantification of SLG in BMDMs treated with DiFMOC-G (0.4 μM) or BrBzGCp2 (5 μM) for 24 h. f Schematic outlines of nucleophilic substitution reaction between SLG and a lysine residue. g Schematic diagram of the three isomer modifications and their different metabolic derivation: KL-La, KD-La, and KCE. h Immunoblot of KD-La levels in innate immune cells stimulated as indicated for 12 h. i Diagram of antibody-enriched lacK peptide identification by LC–MS/MS in BMDMs (left) and fold change of the identified lacK peptides by VSV activation. n = 3 (right). j, k Immunoblot of KD-La levels in BSA (j) or macrophage lysate (k) denatured by boiling or not, then co-incubated with indicated concentration of SLG for 4 h at 37 °C. l Immunoblot of KD-La levels in BMDMs pretreated with indicated concentrations of DiFMOC-G for 24 h. m Immunoblot of KD-La levels in BMDMs pretreated with indicated concentrations of BrBzGCP2 then stimulated by VSV or not. n Immunoblot detection of KD-La levels in Hagh+/+ and Hagh−/− BMDMs. o, p Immunoblot of KD-La levels in BMDMs from WT and GLO2-overexpression (GLO2 OE) mice stimulated by VSV (o) or LPS (p) for indicated time points.
