Figure EV4. Histone lactylation in CD4⁺ T cells.

1. Global histone (Kla) and specific H3K18 lactylation were analyzed by immunoblotting 24 h after stimulation of nonpolarized macrophages. Bone marrow‐derived macrophages were cultured in the presence or absence of 25 mM glucose and 25 mM Na L‐lactate. Immunoblotting of representative whole‐cell extracts is shown (n = 3 biological replicates).
2. Histone preparation by acid extraction from the whole‐cell lysate of murine CD4⁺ T cells, visualized by Coomassie blue staining.
3. Western blots of acid‐extracted histones from activated CD4⁺ T cells showing global histone lactylation in the presence of glucose (25 mM) at 24 h after stimulation of cells. One of three similar experiments is shown.
4. Immunoblotting of acid‐extracted lactylated histones from differentiated Th1, Th17, and Treg cells on day 3 of differentiation. As control lymphocytes, ex vivo purified, nonactivated CD4⁺ T cells were used.
5. ChIP analysis of H3K18‐lactylated histones at the Arg1 and Foxp3 promoter regions in the absence or presence of extracellular lactate (25 mM) was performed after 24 h of the cell culture for Th17 cells. Three independent experiment (n = 3 biological replicates) are shown (n1, n2, n3).

Source data are available online for this figure.