Fig. 3: Acetylation study of murine CD4 T cells.

a, Experimental workflow for the acetylation study of murine CD4 T cells. Lymph nodes and spleen were collected from mice and processed to obtain a single-cell suspension, from which CD4 T cells were isolated. The isolated CD4 T cells were then activated with CD3 and CD28 and treated with lactate or left untreated for 12, 24 and 48 h. The cells were then used to perform a protein acetylation array to assess abundance and levels of acetylation. b, Bar graph listing the number of antibodies with differential intensities when comparing untreated versus lactate-treated (12 h, 24 h and 48 h) CD4 T cells. For each comparison, red indicates upregulated proteins, while blue indicates downregulated proteins. c, Overview of differences in protein abundance and acetylation levels between untreated and lactate-treated (24 h) CD4 T cells. FC, fold change. d, Overview of differences in STAT1 abundance and acetylation levels between untreated and lactate-treated (12 h, 24 h and 48 h) CD4 T cells. e, Relative IL-21 levels in untreated and lactate-treated (12 h, 24 h and 48 h) CD4 T cells. For analysis of the samples, a one-factorial linear model was fitted with LIMMA, resulting in a two-sided t-test (*P < 0.05). Each sample was measured by four replicate spots per array (n = 3 biological replicates). f, Western blot analysis showing the levels of pStat3 and total Stat3 in CD4 T cells. The cells were either left untreated or treated with lactate for 24 h. Data are presented as mean values; error bars, s.d. Statistical significance was assessed using the Mann–Whitney U-test (*P < 0.05, n = 6 biological replicates). g, Selected KEGG pathways related to proteins with differential abundance and acetylation in untreated versus lactate-treated (24 h) CD4 T cells.