Fig. 4. APC-cluster formation in MDCK cells is inhibited by expression of N3 or by the inhibition of 14-3-3 proteins. (A) Representative images of the different types of endogenous APC (a-c) or GFP-APC protein (a'-c') clusters used to categorise cells: (a) cluster, (b) dispersed cluster, (c) dispersed. APC is green; tubulin is shown in red. (B) APC-cluster formation in cells was scored using a total of 100 cells randomly selected from three independent experiments. (a) The localisation of endogenous APC in MDCK cells was scored in cells transiently transfected with N3, C3 or GFP. Expression of N3 caused a marked decrease in the number of cells with clustered APC and led to more cells with dispersed APC, whereas C3 had a more moderate effect. (b) The localisation of GFP-tagged full-length APC in MDCK cells was compared in cells co-transfected with GST or with GST-N3. Again, the presence of N3 led to a decrease in clustering of GFP-APC. Similar data was obtained in PTK2 cells (supplementary material Fig. S4). (c) The localisation of endogenous APC was scored in cells treated with a phospho-peptide (inhibitor) or the non-phospho version of the same peptide (control). Inhibition of 14-3-3 caused a reduction in the number of cells with clustered APC. (d) Clusters formed by GFP-tagged APC lacking N3 (GFP-APCN3) and GFP-APC were scored as above. GFP-APCN3 forms tighter clusters compared with GFP-APC. Similar data was obtained in PTK2 cells (supplementary material Fig. S4). (C) Images of live cells expressing GFP-APC together with mCherry-N3 (a) or mCherry-C3 (b) reveal enrichment of N3 in APC clusters. C3 is not enriched in the clusters, but is also not excluded from them.