Extended Data Fig. 7: Functional characterization of the site-specific cGAS lactylation in cells and lactyl-mimicking or lactyl-destructing knock-in mutations in mice.
From: AARS1 and AARS2 sense l-lactate to regulate cGAS as global lysine lactyltransferases
a, A schematic depicting the engineering strategy for site-specific lactylation of h- and m-cGAS in cells using aminoacyl-tRNA synthetases and aminoacyl tRNA. b, Immunoblot (IB) of the TCLs of engineered cells as described in a. c, Immunoblot (IB) of the TCLs and streptavidin Biotin-DNA (HSV60) pull-down derived from HEK293T cells transfected to express indicated proteins. d-e, In situ PLA of HeLa cells engineered to express indicated proteins and stimulated with HT-DNA for 8 h. f-g, Immunofluorescence and DAPI staining of HeLa cells engineered to express indicated proteins, followed by HT-DNA stimulation for 8 h. h, ELISA of cGAMP in HEK293T cells engineered to express h-cGASNon-lac or h-cGASK131-lac (left), m-cGASNon-lac or m-cGASK156-lac (right) proteins as in a, followed by non-stimulation or HT-DNA stimulation for 8 h. i, Schematic diagram of CRIPSR/Cas9-mediated genome editing of the mouse cGAS gene locus. j, ELISA of IFN-β (left) and plaque assay of HSV-1 titers (right) in the serum of cGASwt/wt and cGASKQ/KQ mice (n = 5) treated with HSV-1 infection. k, Hematoxylin-and-eosin (H&E)-stained lung sections from mice (n = 3) as in i. l, ELISA of IFN-β (left) and plaque assay of HSV-1 titers (right) in the serum of cGASwt/wt and cGASKR/KR mice (n = 5) pre-educated with NaCl (1 g/kg; i.p.) or NaLac (1 g/kg; i.p.) every day for 10 days, followed by HSV-1 infection. m, Hematoxylin-and-eosin (H&E)-stained lung sections from mice (n = 3) as in l. n, ELISA of cGAMP (left), Ifnb1 mRNA (middle) and HSV-1 titer (right) in cGASwt/wt and cGASKQ/KQ peritoneal macrophages by mock infection or infection for 12 h with HSV-1 (MOI, 10). o, ELISA of cGAMP (left), Ifnb1 mRNA (middle) and HSV-1 titer (right) in cGASwt/wt and cGASKR/KR peritoneal macrophages pre-treated for 12 h with NaCl (25 mM) or NaLac (25 mM), followed by mock infection or infection for 12 h with HSV-1 (MOI, 10). The ratio of transfected cGASNon-Lac: cGASLac is 1:4 to ensure equal amount of protein expression. Data are representative of at least three independent experiments (b-h, j-o). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (h, j, l, n, o). Scale bar, 5 μm (d-g). 100 μm (k, m). Supplementary Fig. 1 shows full gels.
