Figure 5.

IVM suppresses Wnt/β-catenin signaling via binding to TELO2

(A and B) Reconstitution of the TELO2 restored β-catenin levels in TELO2-knockdown cells. HEK293 cells were transfected with TELO2 siRNA for 4 days and transfected with an siRNA-resistant FLAG-tagged WT TELO2 expression vector. The cells were treated with 10 μM IVM for 1 h and then with 50 ng/mL Wnt3A for 2 h in the presence of 25 μM MG132. (A) Cell lysates were analyzed through western blotting with anti-β-catenin, anti-TELO2, anti-FLAG, and anti-actin antibodies. The open triangle indicates the bands corresponding to ubiquitinated β-catenin. (B) Band intensities of TELO2 and β-catenin were quantified in control siRNA-transfected (circles), TELO2 #1 siRNA-transfected (triangles), or TELO2-reconstituted (squares) cells in the absence of IVM, normalized to the actin levels, and indicated as values relative to the control siRNA-transfected cells. The X- and Y axes of the left panel indicate relative TELO2 and β-catenin levels, respectively (n = 4 biological replicates). Correlation coefficient (r) = 0.85. Data of the right panels represent the means ± SDs (n = 4 biological replicates). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with Tukey’s test.

(C) TELO2 K749T reconstitution conferred IVM resistance. TELO2-knockdown HEK293 cells were transfected with vectors expressing siRNA-resistant FLAG-tagged WT TELO2 or TELO2 K749T. The cells were treated with 25 μM MG132 for 15 min, 10 μM IVM for 1 h, and 50 ng/mL Wnt3A for 2 h in 1% FBS/DMEM. Protein levels were analyzed through western blotting with specific antibodies (the left panel). The open triangle indicates bands corresponding to ubiquitinated β-catenin. The band intensities were quantified, normalized to actin levels, and indicated as values relative to the control (DMSO) (the right panels). Data are presented as the means ± SDs (n = 3 biological replicates). ∗∗p < 0.01, n.s.: not significant, Welch's t-test. See also .Figure S5