Extended Data Fig. 8: Lactylated cGAS failed to recognize cytosolic mtDNA and self-DNA in Trex1 knock-out mice.

a, Partial Least-squares discrimination analysis (PLS-DA) of the serum metabolome of normal (n = 6, 0–2 mM), high-lac (n = 4, 2–6 mM) and lac-acidosis (n = 2, above 6 mM) patients with HCMV infection. Each symbol represents the data of an individual patients. b, Gene signatures indicating ATP metabolic process (left) or mitochondrial-ATP coupled electron transport (right) are significantly enriched in normal people versus lac-acidosis patients (left) or high-lac patients (right); shown by preranked gene set enrichment analysis (GSEA) of RNA-seq analysis of PBMCs from patients. c, Left: measurement of ATP levels in PBMC of normal (n = 7, 0–2 mM), high-lac (n = 16, 2–6 mM) and lac-acidosis (n = 5, above 6 mM) patients with HCMV infection. Right: mitochondrial DNA (mtDNA) levels in cytosolic fractions from above samples assessed by qPCR amplification of mitochondrial CYTB. d, Fluorescence of GFP fused h-cGAS^Non-lac or h-cGAS^K131-lac and mtDNA (5% labeled with AF647) mixed at room temperature with 150 mM NaCl at pH 7.0. e, Fluorescence of non-lactylated and Lys156-lactylated m-cGAS-GFP mixing with mtDNA (5% labeled with AF647) at room temperature with 150 mM NaCl at pH 7.0. f, Cytosolic mtDNA assessment by qPCR amplification of mitochondrial CYTB from U2OS cells stimulated with either bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES, 30 μM) for 6 h, Doxorubicin (Dox, 500 nM) for 24 h or 5-Fluorouracil (5-FU, 5 μM) for 16 h. g, Mitotracker and Picogreen staining of U2OS cells with PBS or BPTES (30 μM) stimulation for 6 h. h, Immunofluorescence, Picogreen and DAPI staining of U2OS cells engineered to express wt and Lys156-specifically-lactylated m-cGAS, followed by stimulation with BPTES (30 μM) for 6 h. i, Data represent the fold change in enrichment of mtDNA fragments using anti-cGAS to co-precipitate DNA from THP1 cells transfected with control siRNA (Co.si) or si-AARS2, followed by treatment with NaLac (25 mM) for 12 h and BPTES (30 μM) for 6 h as indicated. DNA fragments were amplified by real-time qPCR using three primer pairs (ND1, DLOOP and CYTB) for mtDNA and two primer pairs (L1ORF1, POLG1) for gDNA. j, Data represent the fold change in enrichment of mtDNA fragments using anti-Flag to coprecipitate DNA in HEK293T cells engineered to express wt and site-specifically-lactylated h-cGAS, followed by stimulation with BPTES (30 μM) for 6 h. The quantification of DNA fragments amplified by real-time qPCR using primer pairs (ND1) for mtDNA and primer pairs (L1ORF1) for gDNA were also shown in Fig. 5f. k, Data represent the fold change in enrichment of mtDNA fragments using anti-Flag to coprecipitate DNA in HEK293T cells engineered to express wt and site-specifically-lactylated m-cGAS (K156-Lac), followed by stimulation with BPTES (30 μM) for 6 h. DNA fragments were amplified by real-time qPCR using three primer pairs (ND1, DLOOP and CYTB) for mtDNA and two primer pairs (L1ORF1, POLG1) for gDNA. l, Immunofluorescence and DAPI staining of U2OS cells engineered to express wt and Lys131-specifically-lactylated h-cGAS, followed by stimulation with H₂O₂ (10 mM) for 30 min (left). Quantitative line profile of co-localization along the indicated white arrow of the left image (right). n = 3. m, Representative image (left) and cell number (right) of spleens from Trex1^−/−cGAS^wt/wt and Trex1^−/−cGAS^KQ/KQ mice (n = 10). n, Representative image (left) and histology score (right) of hematoxylin-and-eosin (H&E)-stained heart sections from mice as in m. Scale bar, 100 µm. o, Representative image (left) and histology score (right) of hematoxylin-and-eosin (H&E)-stained skin sections from mice as in m. Scale bar, 100 µm. p, Quantification by RT-PCR of a panel of IFN-stimulated gene transcripts in PBMC (left) or heart (right) from Trex1^−/−cGAS^wt/wt and Trex1^−/−cGAS^KQ/KQ mice (n = 4). q, ELISA analysis of TNF-α, CCL5, CXCL10 and IFN-β in the serum from mice (n = 10) as in m. r, High intracellular L-lactate or AARS2 activity promotes cGAS lactylation thereby severely reducing self-DNA detection and down-regulating the autoimmune surveillance. Data are representative of at least three independent experiments (c-q). Scale bar, 5 μm (d, e, g, h, l). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c, f, i, j-q). Supplementary Fig. 1 shows full gels.

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