Figure 4.

Wnt responses after modulation of the extrinsic (A–E) and intrinsic (H–N) signaling level. (A) Growth of normal colon organoids after titration of Wnt-conditioned medium. In each passage, the mean ATP level was measured (± SD; n = 3 technical replicates; donor #3). Splitting factor was 1:5. Experiment was repeated twice with similar results. (B) Morphological images of organoids after culture at different Wnt concentrations. Scale bar is 1 mm. (C) Strategy to study maximal and submaximal Wnt stimulation. (D) PCA shows dose-dependent changes in WT cells and separate clustering from APC-KO cells (n = 3 technical replicates each). (E and F) Venn diagrams of regulated transcripts (± 1 log twofold change; P adjust < 0.05; E) and global correlation of transcriptomic responses after maximal and submaximal Wnt stimulation (F). (G) GSEA shows incremental induction of the Wnt-receptor signature at distinct levels of receptor stimulation in WT cells. (H) Schematic representation of the APC protein and truncated variants containing three or two 20-aa repeat regions (20AAR, blue). The mutation cluster region (MCR) is indicated. (I) WB analysis of APC (and ACTIN for normalization) in whole-cell lysates of WT and CRISPR/Cas9 induced clonal lines (donor #3). Black and white arrowheads show WT and truncated proteins, respectively. High frequency of a single mutant allele and absence of WT allele was measured by ICE assay. WB and ICE analyses were repeated twice independently. (J) Strategy to study the influence of different APC truncations. (K) PCA shows separate clustering of normal and APC-KO organoids (n = 3 each). Stimulation was performed as in Fig. 1. (L and M) Similar intrinsic response by APC variants with two and three 20AARs. Venn diagrams of regulated transcripts (L; ± 0.5 log twofold change; P adjust < 0.05) and global correlation of changes (M). (N) GSEA shows similar induction of the APC-KO signature by both allelic variants.