Fig. 7: Inhibition of NF-YA by IL-6-activated ERK accounts for the reduction of ACLY ubiquitination and then Th17 differentiation.

a IL-6-triggered-ERK reduces the transcriptional activity of Klhl25 promoter. HEK293T cells were transfected with pGL 4.0-Klhl25 and treated with IL-6 and (or) TGFβ1 in the presence (or absence) of ERK inhibitor (15 μM) before dual luciferase reporter assay. b,c ERK but not STAT3 inhibition rescues Klhl25 mRNA and protein level. Naïve CD4⁺ T cells were cultured under iTreg or Th17 polarization conditions for 48 h in the presence (or absence) of ERK inhibitor and STAT3 inhibitor (10 μM). The mRNA level of Klhl25 was assayed by qPCR (b) and the protein level was detected by WB (c). d IL-6-triggered-ERK inhibits the association of ACLY with CUL3-KLHL25 during human Th17 differentiation. Naive CD4⁺ T cells isolated from human peripheral blood were cultured under iTreg or Th17 polarization conditions for 48 h in the presence (or absence) of ERK inhibitor. Cells were immunoprecipitated with antibodies against ACLY for WB analysis. IgG serves as a negative control. e,f Knockdown of Nfya abolishes the effect of ERK inhibition on the transcription and expression of Klhl25. Naive CD4⁺ T cells transfected with Nfya targeting siRNAs were cultured under iTreg or Th17 polarization conditions in the presence (or absence) of ERK inhibitor and subsequently subjected to qPCR (e) or WB (f) analysis. g Knockdown of Nfya abolishes the effect of ERK inhibition on ACLY ubiquitination. Cells transfected and cultured as described in the legend to (e, f) were treated with MG132 before immunoprecipitated with antibodies against ACLY (g) or Ub (h) for WB analysis. IgG serves as a negative control. h,i Knockdown of Nfya abolishes the effect of ERK inhibition on FAO and Th17 differentiation. h Cells were cultured as described in the legend to (e, f). For oxygen consumption rate (OCR) detection, cells were transferred to XF Base Medium containing palmitate and L-carnitine. Diagram (up) illustrating the OCR at various conditions and associated quantifications (down) are shown. i Cells cultured as described in the legend to (e, f) for 72 h were analyzed by flow cytometry (FCM) for the differentiation of CD4⁺ IL-17A⁺Th17 (left) and quantified (right). j Knockdown of Klhl25 abolishes the effect of ERK inhibition on Th17 differentiation. Naive CD4⁺ T cells transfected with Klhl25 targeting siRNAs were cultured under iTreg or Th17 polarization conditions in the presence (or absence) of ERK inhibitor and subsequently analyzed by FCM for the differentiation of CD4⁺ IL-17A⁺Th17 (left) and quantified (right). For (a–j), data are representative as mean ± SD (n = 3 biologically independent samples) with p values determined by one-way ANOVA test. For WB in (c, d, f, g), one representative experiment out of three is represented. The values indicate mean intensities based on three biological replicas.