Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner (original) (raw)
Figure 6
Wnt signalling is active in naïve CD4+ T cells and differentiating TH cells.
(A) Naïve CD4+ T cells were isolated from cord blood as described in Materials and Methods. Wnt signalling activity was measured by performing transactivation assay using TOPFlash and FOPFlash reporter constructs. TCF reporter activity was measured after 48 h in untreated control cells (bar 1), cells treated with Wnt inhibitor Dkk1 (bar 2), and upon cotransfections of si-β-catenin (bar 3) and β-catenin (T41A) (bar 4) as indicated. In all samples the reporter activity was measured without adding any Wnt agonist. The ratio of luciferase activities in TOPFlash transfected versus FOPFlash-transfected cells was determined and plotted as the relative TCF activity. Each error bar indicates standard deviation calculated from triplicates. (B) Expression of Wnts in CD4+ T cells. Naïve CD4+ T cells were isolated from cord blood as described in Materials and Methods. The mRNA levels of indicated Wnts were determined by RT-PCR analysis of total RNA extracted from these cells (top panel). Wnt expression was also monitored by quantitative RT-PCR analysis of RNAs from two biological replicates (lower graph). Expression level of Wnt 5b was considered as one unit for calculating relative fold expression of other Wnts after normalizing with GAPDH expression in the same sample. (C) Immunoblot analysis was performed to monitor stabilization of β-catenin in differentiating TH cells. Naïve CD4+ cells were activated using plate-bound anti-CD3 and soluble anti-CD28 and differentiated by adding IL-4 or IL-12 as described in Materials and Methods. Nuclear extracts were prepared from control cells and cells polarized to TH1 and TH2 and were treated with Wnt agonist BIO or Wnt inhibitor Dkk1, followed by immunoblot analysis using anti-β-catenin (upper panel) and anti-γ-tubulin (lower panel). Each lane represents protein isolated from 5×106 differentiated and treated cells. The numbers below the β-catenin panel represent fold change in β-catenin expression in the indicated lanes of the immunoblot upon normalization with that of γ-tubulin as loading control. Fold change values were calculated from densitometric quantitation of respective bands. Numbers below the γ-tubulin panel denote individual lanes of the immunoblot.