Fig. 1: ACLY-mediated FAS is crucial for IL-6-induced Th17 differentiation.

Analyses of FAS and FAO in iTreg and Th17. Naïve CD4⁺ T cells were cultured under Th0, iTreg or Th17 polarization conditions for 48 h in the presence of [U-¹³C] glucose (11 mM) (a) or [U-¹³C] palmitate (100 μM) (b). Cells were collected and subjected to metabolic flux analysis for FAS (a) or FAO (b) by UHPLC-HRMS analysis. c,d Assay of the protein and enzymatic activity of ACLY in iTreg and Th17. Cells were cultured as described in the legend to (a) for 72 h and subjected to analyses for ACLY protein level by WB (c) and ACLY enzymatic activity by enzymatic activity assay kit (d). e Knockdown of Acly reduces Th17 differentiation. Naïve CD4⁺ T cells isolated from mice were transfected with negative control (NC) or Acly targeting siRNAs and were cultured as described in the legend to (a) for 72 h. Malonyl-CoA (50 μM) was added (or not) to the culture at the onset. Cells were stained for CD4/IL-17A to identify Th17 prior to flow cytometry (FCM) analysis (left) and quantification (right). f Overexpression of ACLY enhances Th17 differentiation. Cells transfected with pMIG-Acly overexpression virus were polarized as described in the legend to (e) and were stained for CD4⁺IL-17A⁺Th17 analysis by FAM (left) and quantification (right). For (a–f), data are representative as mean ± SD (n = 4 (a) or 3 (b–f) biologically independent samples) with p values determined by one-way ANOVA test. For WB in (c, e, f), one representative experiment out of three is represented. The values indicate mean intensities based on three biological replicas.