Extended Data Fig. 3: Genome-wide CRISPR screen identifies AARS1/2 as intracellular L-lactate sensor.

a, Schematic overview of the GFP reporter construct. GFP expression is driven by interferon-stimulatory response elements (ISRE) (left). HT-1080 cells stably expressing the GFP reporter were incubated with L-lactate (25 mM) for 24 h, followed by HSV-1 infection (MOI, 10) for 12 h. GFP reporter response was analyzed by flow cytometry after the infection (right). b, Distribution of the fold change in the positively selected genes enriched in the GFP-high HT-1080 cells. c, Bacterial purified AARS1-wt, AARS2-wt and AlaRS-wt proteins were analyzed by SDS-PAGE and detected by Coomasssie blue staining. d, Human AARS1 (left), AARS2 (middle) and E. coli AlaRS (right) directly bound alanine and L-lactate in vitro with indicated K_d in Microscale thermophoresis (MST) binding affinity analysis. Data represent mean ± s.d. n = 3 per group. e, Boxplot of global lysine lactylation intensity of samples as in control, AARS1 and/or AARS2 depleted HeLa cells treated without or with L-lactate (Lac, 25 mM) for 24 h. In the box plots, the middle bar represents the median, and the box represents the interquartile range; bars extend to 1.5× the interquartile range. The P values were calculated by the two-sided Wilcoxon rank-sum test. n = 220 proteins. f-g, Scatter plots of global lysine lactylation intensity between indicated samples as in e. h, Sequence motif analysis showed the amino acid residue preference of AARS1 (upper), AARS2 (middle) and AARS1 + AARS2 (lower) around the identified lysine lactylation sites. The red horizontal lines denote thresholds of p < 0.05. These significance values are calculated using the binomial probability of residue frequencies, with respect to a genomic or proteomic background based on pLogo (https://plogo.uconn.edu/). Data are representative of at least three independent experiments (a, c, d). Supplementary Fig. 1 shows full gels.

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