Fig 2: Truncation of APC affects abundance and diffusional dynamics of wild-type but not mutant ß-catenin in the cytosol.(A) Schematic representation of the APC locus and target site of sgACP in the mutation cluster region (MCR) domain. (B) Concentrations (left) and diffusion coefficients (right) of Clover-tagged wild-type ß-catenin and Cherry-tagged mutant ß-catenin in the cytosol of HCT116 ß-cateninWTClover/?45Cherry and sgAPC targeted clone (APCLOF) cells that were treated for ~16 hr with either 10 µM CHIR9901 or DMSO as control. Subsequently, FCS measurements were performed in the cytosol (data shown here) and in the nucleus (data shown in Figure 7—figure supplement 1A). Each data point in the box plots represents a result from a 120 s FCS measurement in a single cell. Per box plot, more than 40 cells were investigated in three independent experiments. p-values were calculated with the Mann-Whitney test (*** <0.001; ** <0.01; * <0.05; NS – non-significant). The exact values are provided in the Supplementary file 1. FCS, fluorescence correlation spectroscopy. Figure 7—source data 1.Truncation of APC affects abundance and diffusional dynamics of wild-type but not mutant ß-catenin in the cytosol.(B) HCT116 ß-cateninWTClover/?45Cherry and sgAPC targeted clone (APCLOF) cells were treated for ~16 hr with either 10 µM CHIR9901 or DMSO as control. Subsequently, FCS measurements were performed in the cytosol (data shown here) and in the nucleus (data shown in Figure 7—figure supplement 1A). Each data point in the box plots represents a result from a 120 s FCS measurement in a single cell. Per box plot, more than 40 cells were investigated in three independent experiments. p-values were calculated with the Mann-Whitney test (*** <0.001; ** <0.01; * <0.05; NS – non-significant). The exact values are provided in the Supplementary file 1. Figure 7—figure supplement 1: Truncation of APC affects abundance and diffusion of wild-type but not mutant ß-catenin in the nucleus. (A) HCT116 ß-cateninWTClover/?45Cherry and sgAPC targeted clone (APCLOF) cells were treated for ~16 hr with either 10 µM CHIR9901 or DMSO as control. Afterward, FCS measurements were performed in the cytosol (data shown in Figure 7) and in the nucleus (shown here). For each data point, a 120 s FCS measurement was carried out in a single cell. Per box plot, more than 40 cells were examined in three independent experiments. p-values were calculated with the Mann-Whitney test (*** <0.001; ** <0.01; * <0.05; NS – non-significant). The exact values are provided as the Supplementary file 1. FCS, fluorescence correlation spectroscopy. Figure 7—source data 2.Truncation of APC affects abundance and diffusion of wild-type but not mutant ß-catenin in the nucleus.Figure 7—figure supplement 1: (B) Both ß-catenin alleles of HCT116 cells bind to APC, GSK3ß, and Axin1. IP with anti-APC antibody was performed GFP/Clover and Cherry were detected (left panel). IPs with RFP/Cherry and GFP/Clover beads were performed and ß-catenin, Axin1, and GSK3ß were detected (right panel). Representative experiments from three independent are shown.

Fig 3: Wnt signaling is dysregulated in inv-/- MEFs.(A) WB analysis of growth arrested inv+/+ and inv-/- MEFs with antibodies against Dishevelled (Dvl) -1, -2 and -3, Adenomatous polyposis coli (APC), Glycogen synthethase kinase 3 ß (GSK3ß), ß-catenin (ß-cat) and S45-phosphorylated ß-catenin (p-ß-cat). (B) Quantifications of WB analysis from (A). Histograms represent mean ± S.E.M. (n=3). (C,G) IFM analysis of growth arrested inv+/+ and inv-/- MEFs. Primary cilia (Ac-tub or Glu-tub, red) are marked with closed arrows, nuclei are stained with DAPI (blue). Green protein localizations are Dvl-1 (C), Dvl-2 (D), Dvl-3 (E), p-ß-cat (F) and Frizzled-3 (Fzd-3) (G). (H,I) IFM analysis on the localization of S33/37-T41-phosphorylated ß-catenin (p-ß-cat) (blue, open arrows) to the ciliary base (asterisks) in mock (H) and Inv::GFP (green) (I) transfected inv+/+ and inv-/- MEFs. Primary cilia (Glu-tub, red) are marked with closed arrows.

Fig 4: Both PKCd and APC bind to the c-Myc enhancer in vivo.Serum starved RKO cells (8 h) were pre-incubated for 2 h in the absence or presence of the PKCd inhibitor rottlerin (6 µM), and then incubated in the absence or presence of Wnt3a conditioned medium during 50, 90 or 120 min. The ChIP analysis of the time course of cell treatment was performed as described in Materials and Methods section. PCRs were done with primers spanning the upstream TCF/LEF sites in the human C-MYC gene enhancer (spanning the region corresponding to -1447 to -1144 relative to the P1 RNA start site +1) as described in (9). PCR of the input DNA prior to immunoprecipitation was used as a control. A) ChIP specificity controls: conditions employed were specific, because TCF4 and calreticulin bound to total chromatin, but calreticulin, a protein not related to Wnt signaling did not bind to the c-Myc enhancer but the TCF4 transcription factor appeared bound to it. Neither calreticulin nor TCF4 were found bound at a distal upstream region (1 kb) of the C-MYC gene (control). B) Time course of RKO cells treatment in the absence or presence of Wnt3a conditioned medium and in the absence or presence of rottlerin. Results shown are representative of three experiments using different cell preparations.

Fig 5: PKCd phosphorylates APC in intact RKO cells. A) Table I. Putative PKC phosphorylation sites in APC (NetphosK 2.0). B) Metabolic labeling and determination of specific phosphorylation (left). Western blot with anti Phospho-Ser-PKC substrate (right). 24 h post-transfection of RKO cells with the pControl (void) or pHA-DNPKCd plasmids, RKO cells were serum-starved (2% instead of 10%) for 4 h, washed and incubated with 150 µCi/ml [32P] Pi in phosphate-free DMEM medium supplemented with 2% FBS for a total period of 4 h. Wnt3a (100 ng/ml, or vehicle) was added during the last hour of incubation period as described in Material and Methods. APC was immunoprecipitated from the corresponding cell lysates, subjected to SDS-PAGE, transferred to nitrocellulose, and processed for autoradiography or for Western blotting. Autoradiography: the determination of protein content was done after radiography in the corresponded dried gel. Autoradiograms and their corresponding dried gels were quantified with an image densitometer and the specific phosphorylation was determined as the ratio of phosphorylated protein to the total protein content. Values plotted are means ± S.E.M. for three experiments performed with different cell preparations. Western blotting: APC immunoprecipitates were analyzed for the presence of APC phosphorylated in Ser residues by PKC. The same blots were erased and reprobed with APC antibodies. Also is shown the HA-tagged kinase-dead PKCd over-expression and actin loading controls. A representative experiment from three experiments performed with different cell preparations is shown.