Figure 4. Wnt Receptor Activation in APC-Deficient Cells Is Wnt Ligand Independent.
(A–D) Fz8-Fc blocks Wnt ligand-mediated signaling but not signaling in APC-deficient cells. (A) RKO STF cells were incubated with Wnt3a and Fz8-Fc at the indicated concentrations. (B) HEK293 STF cells transfected with APC siRNA and (C) RKO APCKO cells were incubated with Fz8-Fc. (D) Wnt activation in CRC cells is not dependent on Wnt ligand. CRC cells were incubated for 48 hr with Fz8-Fc at the indicated concentrations.
(E) APC-depleted cells are responsive to Wnt ligand. RKO APCKO cells were preincubated for 30 min with Fz8-Fc followed by incubation with Wnt3a.
(F) Inhibition of secreted Wnt3a by the PORCN inhibitor Wnt-C59. HEK293 STF were transfected with Wnt3a and treated with Wnt-C59 (0.3 nM).
(G) Wnt pathway activation by exogenous Wnt3a is not blocked by Wnt-C59. HEK293 STF cells were preincubated for 30 min with Wnt-C59 followed by incubation with Wnt3a.
(H and I) PORCN inhibition does not block Wnt activation upon loss of APC. (H) HEK293 STF cells transfected with APC siRNA and (I) RKO APCKO cells were incubated with Wnt-C59.
(J) PORCN inhibition does not block Wnt activation in CRC cells. CRC cells were incubated for 48 hr with Wnt-C59.
(K) LRP6 is required for Wnt activation upon loss of APC in the absence of PORCN. PORCNKO MEF and wild-type MEF were transfected with APC and LRP6 siRNA.
Graphs show mean ± SEM. n.s., non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S3.