Figure 5. Nrf2 is a negative regulator of Wnt signaling dependent on the β-catenin N-terminus.

(A) HEK293T were transfected with TOP or FOP, CMV::Renilla, differing amounts of ΔN89-β-catenin (balanced with empty vector) with either empty vector or Nrf2. The y-axis is in log scale, making blue line appear curved but it best-fits a straight line (linear fit slope: 1500 ± 53, R²=0.995). (B) Data from (A) are shown as the ratio of Wnt signaling for empty vector/Nrf2 and expressed as a function of CMV::ΔN(89)-β-catenin dose. Wnt activity shows a hook/prozone effect upon titrating ΔN89-β-catenin in a Nrf2-overexpressing background. (C) HEK293T cells were transfected with ΔN(89)-β-catenin (Flag-tagged) together with either empty vector or Nrf2 (myc-tagged). After 48 h, levels of ΔN(89)-β-catenin were assessed by anti-Flag IF. (D) Same as (A) but β-catenin-GFP was used. The y-axis is in log scale, making blue line appear curved but it best-fits a straight line. (E) HEK293T cells expressing control shRNA were transfected with the indicated β-catenin construct, TOP or FOP, CMV::Renilla (see inset in Fig. 4A) and varying amounts of Nrf2 plasmid balanced with empty vector. After 48 h, Wnt signaling was assessed. Inset: At 1 equiv Nrf2, β-catenin N-terminal deletion mutants have higher Wnt activity than wt-β-catenin. (F) As in (E), except β-catenin with or without GFP fusion is compared over Nrf2 titration. Inset: HEK293T cells were transfected with TOP or FOP, the indicated β-catenin construct, and CMV::renilla, and Wnt signaling was measured after 48 h. (G) Same as (E) except indicated shRNA knockdown lines were used. Note: Nrf2 titration against wt-β-catenin in (E) is reshown in (F) and (I) for comparison. (H) Same as (E) except the indicated HEK293T lines were used. (I) The indicated HEK293T lines were transfected with wt-β-catenin, TOP or FOP, CMV::Renilla, 0.05 equivalents of Nrf2, and the plasmid encoding the indicated transgene or empty vector, and Wnt activity was measured after 48 h. (see β-TrCP-domain structure in Fig. S5I). Data present Mean ± s.e.m. with each bar graph from n>3 independent biological replicates.