Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 5. Hyperlactylation of Ikzf1 at Lys164 is important for TH17 differentiation. (A) Radar diagram showing the top 15 DLPs in CD4+ T cells of EAU mice. (B) Enriched GO biological processes of up-regulated Kla proteins. (C and D) MS/MS spectra of the lactylated peptides of Ikzf1 at Lys164 and Lys373. (E) Immunoblot after immuno- precipitation (IP) assays showing increased lactylation of Ikzf1 in CD4+ T cells of EAU mice (n = 4 samples per group; **P < 0.01 by two-tailed unpaired Student’s t test). (F) Expression of Ikzf1 in CD4+ T cells of TH0 and TH17 groups measured through Western blotting (n = 4 samples per group; ns, no significance by two-tailed unpaired Student’s t test). (G) CD4+ T cells were transfected with the control virus (control), only sh-Ikzf1 (sh-Ikzf1), shIkzf1+oeWT-Ikzf1 (oeWT-Ikzf1), shIkzf1+oeK164R Ikzf1 (oeK164R-Ikzf1), and shIkzf1+oe K373R Ikzf1 (oeK373R-Ikzf1) adenovirus. Expression levels of Ikzf1 in different groups were measured through Western blotting (n = 4 samples per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (H) FCM analysis of the frequency of TH17 cells in different groups as described in (G) (n = 4 samples per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (I) Expression levels of IL-17 in the media of corresponding groups were tested by ELISA (n = 8 samples per group; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis test). m/z, mass/charge ratio.

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Tandem Mass Spectroscopy, Western Blot, Immunoprecipitation, Two Tailed Test, Expressing, Transfection, Control, Virus, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 6. Ikzf1 K164la was up-regulated in CD4+ T cells of EAU mice. (A) Dot blot assays of Ikzf1-K164la antibody. (B) Ikzf1-K164la levels in TH0 and TH17 cells measured by Western blotting (n = 4 samples per group; **P < 0.01 by two-tailed unpaired Student’s t test). (C) Ikzf1-K164la levels in TH17 cells in response to DCA and rotenone treatment (n = 3 samples per group; *P < 0.05 by one-way ANOVA and Bonferroni post hoc test). (D) Ikzf1-K164la levels in CD4+ T cells of EAU mice at different time points (n = 4 samples per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (E) Ikzf1-K164la levels in CD4+ T cells of EAU mice in response to DCA and rotenone treatment (n = 3 to 4 samples per group; *P < 0.05 by one-way ANOVA and Bonferroni post hoc test).

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Dot Blot, Western Blot, Two Tailed Test

Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 7. CUT& Tag analysis reveals the transcriptional consequences of Ikzf1 under TH17 differentiation condition. (A) Binding density of WT Ikzf1 was visualized by deepTools. The heatmap presents the CUT& Tag counts on the different Ikzf1 binding peaks in CD4+ T cells between WT and K164R groups under TH17 induction con- dition, ordered by signal strength. (B) Genome-wide distribution of Ikzf1 binding peaks in CD4+ T cells of WT and K164R groups. (C) GO analysis of the decreased Ikzf1 binding peaks at candidate target genes. (D) Genome browser tracks of CUT& Tag signal at the representative target gene loci. (E) mRNA expression levels of IL-2, IL-4, Tlr4, and Runx1 measured using RT-qPCR (n = 4 samples per group; **P < 0.01 and ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (F) Luciferase activity of the IL-2 promoter–driven reporter vector was measured between the control, sh-Ikzf1, shIkzf1+oeWT-Ikzf1, shIkzf1+oeK164R-Ikzf1, and shIkzf1+oe K373R Ikzf1 groups (n = 8 samples per group; ***P < 0.001 by Kruskal-Wallis test). (G) Luciferase activity of the Runx1 promoter–driven reporter vector was measured between the control, sh-Ikzf1, shIkzf1+oeWT-Ikzf1, shIkzf1+oeK164R-Ikzf1, and shIkzf1+oe K373R Ikzf1 groups (n = 8 samples per group; ***P < 0.001 by one-way ANOVA and Dunnett’s T3 post hoc test). (H) FCM analysis of the frequency of TH17 cells in corresponding groups (n = 4 per group; ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). (I) Ex- pression levels of IL-17 in the media of corresponding groups tested by ELISA (n = 8 per group; *P < 0.05 and ***P < 0.001 by one-way ANOVA and Bonferroni post hoc test). 50UTR, 50 untranslated region.

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Binding Assay, Genome Wide, Expressing, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation, Control, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Global lactylome reveals lactylation-dependent mechanisms underlying T H 17 differentiation in experimental autoimmune uveitis.

doi: 10.1126/sciadv.adh4655

Figure Lengend Snippet: Fig. 8. Schematic diagram of the current study. Global lactylome reveals that Ikzf1 lactylation levels are up-regulated in the CD4+ T cells of EAU mice. Further exper- iments demonstrated that Ikzf1 K164 lactylation promotes TH17 differentiation by regulating IL-2, IL-4, Tlr4, and Runx1 expression.

Article Snippet: The following primary antibodies were used: Pan-Kla (diluted 1:1000; PTM1401RM), Ikzf1-K164la (PTM), Ikzf1 (diluted 1:1000; 14859, CST), β-actin (diluted 1:3000; Proteintech, 20536-1-AP), and FLAG (diluted 1:1000; 14793, CST).

Techniques: Expressing