Extended Data Fig. 2: Myeloid MCT1 mediates the exogenous L-lactate-induced cGAS inactivation and the lactylated cGAS are enzymatically much less active.
From: AARS1 and AARS2 sense l-lactate to regulate cGAS as global lysine lactyltransferases
a, ELISA of cGAMP (left), qPCR analysis of Ifnb1 mRNA, and plaque assay of the HSV-1 titer (right) in Lyz2-Cre−Mct1f/f and Lyz2-Cre+Mct1f/f peritoneal macrophages pre-treated for 24 h with NaCl (25 mM) or NaLac (25 mM), followed by infection with mock (PBS) or HSV-1 (MOI, 10) for 12 h. b, Immunoblot (IB) analysis of monomeric (Mono) and dimeric (Dimer) IRF3 (top blot; native PAGE) and total lysates (bottom blot; denaturing gel) of Lyz2-Cre−Mct1f/f and Lyz2-Cre+Mct1f/f peritoneal macrophages pre-treated with (+) or without (−) L-lactate (25 mM) for 12 h, followed by mock infected (PBS) or infection for 12 h with HSV-1 (+). c, Lyz2-Cre−Mct1f/f and Lyz2-Cre+Mct1f/f mice (n = 5) were pre-educated with sodium chloride (NaCl) (1 g/kg; i.p.) or sodium L-lactate (NaLac) (1 g/kg; i.p.) every day for 10 days, followed by HSV-1 infection for 24 h. d, ELISA of cGAMP (left) and IFN-β (right) in serum from mice as in c. e, ELISA of cGAMP (left), qPCR analysis of Ifnb1 mRNA (right) in the lung, spleen and liver of Lyz2-Cre−Mct1f/f and Lyz2-Cre+Mct1f/f mice (n = 5 animals per group) as in c. f, Plaque assay of HSV-1 titer in lung, liver, and spleen of infected mice as in c. g, qPCR analysis of HSV-1-specific genomic DNA (gDNA) in the lung, spleen and liver of mice as in c. h, Immunoblot of HSV-1 (UL46) in lung, liver, and spleen of infected mice as in c. i, Survival of mice (n = 10 animals per group) as in c at various days after infection with HSV-1. j, ELISA of the in vitro cGAMP synthesis (left) by incubating Recombinant (Rec.) human (h-) or mouse (m-) cGAS, ATP (2 mM), GTP (2 mM), HT-DNA (0.01 mg/ml) with mock (PBS), NaCl (2 mM), NaLac (2 mM) and lysate of HEK293T cells as indicated in the left scheme, cell lysate for the 5th and 7th reactions were preheated at 95 °C for 5 min to denature proteins (left). Immunoblot (IB) of pan-Klac and the cGAS input were shown (right). k, Measurement of intracellular L-lactate in Lyz2-Cre−Mct1f/f and Lyz2-Cre+Mct1f/f peritoneal macrophages after incubation with NaCl (25 mM) or NaLac (25 mM) for indicate time points. l, m, ELISA of the in vitro cGAMP synthesis as in j using h- or m-cGAS purified from HEK293T (l) or RAW264.7 cells (m) pre-treated with PBS, L-lactate (25 mM), or NaLac (25 mM) for 24 h. Scheme of purification (left), immunoblot (IB) verification of the Flag-tagged and lactylated h- and m- cGAS (middle) and ELISA of cGAMP (right) were shown. All data are representative of at least three independent experiments. Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (a, d-g, j-m), or log-rank test (i). Supplementary Fig. 1 shows full gels.
