Supplementary Figure 6: High-salt-induced β-catenin activation via AKT is independent of Wnt ligands.
From: Activated β-catenin in Foxp3+ regulatory T cells links inflammatory environments to autoimmunity
(a) Relative frequency of IFN-γ and IL-10 positive cell number (fold of control condition) in human Treg cells. Treg cells were stimulated with anti-CD3 and anti-CD28 in the presence of Fzd8-FC (Fzd), additional 40 mM NaCl (NaCl), or Fzd8-FC and NaCl (NaCl + Fzd) (n = 7 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (b) Relative expression level of active β-catenin in human Treg cells cultured as in (a). (n = 7 subjects) *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (c) GSEA enrichment plots of PI3K/AKT pathway gene sets between IFN-γSP vs. IL10SP. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated at the bottom of each plot. (d) Relative expression level of p-AKT on Treg subsets. Treg cells were stimulated with anti-CD3 and anti-CD28 for 4 days, followed by 4 h PMA/iomomycin stimulation and intracellular cytokine staining for IFN-γ and IL-10 (n = 5 subjects). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). (e) Flow cytometric analysis of β-catenin phosphorylation at s522 in human Jurkat T cells. Human Jurkat T cells were stimulated in the presence of AKT inhibitor MK2206 (AKT-i), additional 40 mM NaCl (NaCl), or MK2206 and NaCl (NaCl + AKT-i) (n = 4). (f) Flow cytometric analysis of GSK3β phosphorylation at s9 and AKT phosphorylation at s473 in human Jurkat T cells. Human Jurkat T cells were prepared as in Supplementary Fig. 6c. (n = 4). *P < 0.05, **P < 0.01 (one-way ANOVA with Tukey’s multiple comparisons test). Data were represented as mean + /- SD.
