Figure 2.

Twa1 promotes β-catenin nuclear accumulation and target gene expression in response to Wnt signaling. HEK-293 cells treated with lentivirus-based shRNAs targeting different regions of Twa1 mRNA (sh-Twa1 and sh-Twa1-2), β-catenin mRNA (sh-β-catenin) or control shRNA (sh-ctr) were transfected with an RNAi-resistant human Twa1 (Twa1^*) construct or not. The cells were then treated with Wnt3a-conditioned medium (Wnt3a-CM) or control medium (Ctr-CM), and subjected to the following analyses. (A-D) Effect of Twa1 depletion on Wnt-induced luciferase reporter activity (A, C) or Wnt target gene expression (B, D). Quantitative data are expressed as the mean ± SEM (at least three independent experiments). ^*P < 0.05 and ^**P < 0.01, Student's t-test. (E) Western analysis of endogenous β-catenin and Twa1 from cytosolic and nuclear fractions of HEK-293 cells. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (F, G) Confocal microscopy of β-catenin nuclear localization. Green signals represent cells infected with lentiviruses. DNA was visualized with DAPI (blue). The intensity of red signals in the nuclei of GFP-positive cells was plotted. The nuclear intensity in the GFP-positive cells treated with both sh-ctr and Wnt3a-CM is set as 100%. More than 50 nuclei are measured in each group. Scale bars, 10 μm. ^**P < 0.01, Student's t-test. (H) Co-IP analysis with the indicated antibodies showing the interaction between endogenous β-catenin and TCF4. (I) Western analysis with the indicated antibodies showing the effect of β-catenin depletion on Twa1 nuclear accumulation upon Wnt3a stimulation. IB, immunoblotting; IP, immunoprecipitation.