Fig 2: CUL3-KLHL25-ACLY ubiquitination axis regulates human iTreg differentiation.(A) Assay of ATP-citrate lyase (ACLY) expression in human inducible regulatory T (iTreg) cells. Naive CD4+ T cells isolated from human peripheral blood were cultured with Dynabeads Human T-Activator CD3/CD28 and rhIL-2 to induce activated T (Th0) cells or simultaneously supplemented with rhTGFß1 (2 ng/ml) to induce iTreg cells for 24 hr. Cell extracts were subsequently analyzed for ACLY expression by western blotting (WB). (B) ACLY overexpression affects iTreg cell differentiation. Human naive CD4+ T cells transfected with GFP-ACLYWT were polarized as in (A) for 72 hr prior to the assessment of iTreg cells by flow cytometry (FCM) (left). Quantification of iTreg populations (right). (C) ACLY inhibition affects iTreg differentiation. Cells treated with SB204990 were cultured under Th0- or iTreg (with 0.5 ng/ml rhTGFß1)-polarization condition for 72 hr and subjected to the analysis (left) and quantification (right) of iTreg cells. (D) ACLY interaction with CUL3-KLHL25. Cells prepared as in (A) were pre-treated with MG132 before immunoprecipitation (IP) with ACLY antibody. IgG serves as a negative control. (E–G) ACLY ubiquitination regulates human iTreg differentiation. Cells transfected with GFP-ACLYWT or -ACLY3KR were induced under Th0- or iTreg-polarization condition as described in (A) for 24 hr (E, F) or 72 hr (G). Cells were pre-treated with MG132 before IP for the assessment of protein ubiquitination (E), examined by WB (F), or evaluated for GFP/CD25/Foxp3 expression with FCM (left), and quantified (right) (G). (A–C, F, G) Quantification shows mean ± SD based on three independent experiments, with significance determined by Student's t-test (A, C) or one-way analysis of variance (ANOVA) test (B, F, G). *p<0.05, **p<0.01, ***p<0.001; ns, nonsignificant. For WB in (A) and (D–F), one representative experiment out of three is represented. Associated scores indicate mean intensities based on three biological replicas.