APC Inhibits Ligand-Independent Wnt Signaling by the Clathrin Endocytic Pathway

Cell Lines

The following cells were generous gifts: HEK293 STF (J. Nathans, Johns Hopkins University), RPE (I. Kaverina, Vanderbilt University), MEF and MEF PORCN^KO (C. Murtaugh, University of Utah), 293FT (D. Carbone, Ohio State University), HEK293T Dvl TKO (S. Angers, University of Toronto), HCT116 WT KO (B. Vogelstein, Johns Hopkins University), and MEF LRP6^flox (B. Williams, Van Andel Institute). HEK293, RKO, SW480, DLD1, L-Wnt3a, L-cells, and MEF Caveolin-1^KO were purchased from the American Type Culture Collection. HEK293 CMV-luc was reported previously (Thorne et al., 2010). Cell lines were maintained in DMEM except for SW480 and DLD1 cells (RPMI) and HCT116 (McCoy’s) with 10% FBS and 1% penicillin/streptomycin at 37°C in 5%CO₂.

HEK293 cells were derived from human embryonic kidney cells obtained from a healthy fetus. RPE (hTERT-RPE1) cells were isolated from a 1-year-old female donor (Rambhatla et al., 2002). DLD1 cells were obtained from a 45-year-old male donor. HCT116 cells were obtained from a 48-year-old male donor. SW480 cells were obtained from a 50-year-old male donor (Ahmed et al., 2013). L cells were obtained from 100-day old male mice. MEF Caveolin-1^KO cells were obtained from E13.5 mouse embryos. RKO, MEF, MEF PORCN^KO, and MEF LRP6^flox cells were described previously (Ahmed et al., 2013; Barrott et al., 2011; Zhong et al., 2012).

Three-Dimensional Organoid Culture

Organoids from isolated polyps of APC^min mice were prepared as previously described (Li et al., 2017). Jejunum from ~8-week-old APC^min mice (C57BL/6J-Apc^Min/J) was obtained and washed with phosphate-buffered saline (PBS) followed by incubation for 20 min with cold PBS containing 1.5 mM dithiothreitol and 30 mM EDTA. Tissue was then incubated with warm PBS containing 15 mM EDTA for 6 min, vigorously shaken to release intestinal crypts, and centrifuged. The resulting crypt pellet was washed with 30× volume of basal media (Dulbecco’s modified Eagle media/F12 plus 2 mM GlutaMAX, 10 mM Hepes, penicillin/streptomycin (100 U/mL), and 1× N2 and 1× B27 supplements). Purified crypts were filtered with a 100 µm cell strainer and embedded in Matrigel (Corning). Isolated organoids were maintained in basal media supplemented with niche factors (epidermal growth factor (50ng/mL), R-spondin1 (250ng/mL), Noggin (100ng/mL), and 1mM Jagged-1) with or without Wnt3a (using 25% L-WRN conditioned media (Miyoshi and Stappenbeck, 2013)) and Pitstop-2 or mAb7E5 for 4 days. All experiment procedures were carried out in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and with the policies of the University of Miami IACUC.

Drosophila Stocks

RNAi lines were expressed in progenitor cells using temperature-sensitive progenitor cell driver esg^ts (UAS-dicer2; esg-gal4 tubgal80^ts UAS-GFP/CyO) (Guo et al., 2013; Micchelli and Perrimon, 2006):

Apc1 RNAi (Vienna Drosophila Resource Center [VDRC] #51469), arr RNAi lines (arrⁱ¹, VDRC#4819; arrⁱ², VDRC #6707; arrⁱ³, Bloomington Drosophila Stock Center [BDSC] #53342 and arrⁱ⁴, VDRC #6708), dsh RNAi lines (dshⁱ¹, BDSC #31306; dshⁱ², VDRC #101525), wg RNAi lines (wgⁱ¹, VDRC #104579; wgⁱ², VDRC #13351), wls RNAi (VDRC #103812) and y RNAi (BDSC #64527).

Nkd-lacZ (Zeng et al., 2000) was used as Wg target gene reporter.

Apc1^Q8 (Ahmed et al., 1998) was used as a null allele.

Transfections

Plasmid transfections were performed using CaCl₂, Lipofectamine 3000 (Invitrogen), or FuGene HD (PROMEGA). CaCl₂ transfections were performed as follow: cells were seeded at 50%–60% confluency in 6-well plates and incubated overnight. To transfect an individual well: 1µg of DNA was mixed with 6.1µl of CaCl₂ and sterile water for a final concentration of 100 µl, and added to a tube containing 100 µl of 2× HBS (280 mM NaCl, 10mM KCl, 1.5mM Na₂HPO₄-2H₂O, 12mM Dextrose (D-Glucose), and 50mM HEPES). Mixed by bubbling air and added dropwise to the cells. Lipofectamine 3000 and FuGene transfections according to manufacturer’s protocol. siRNA transfections were performed using Dharmafect1 (HEK293 and RKO cells), Dharmafect2 (SW480, DLD1, and HCT116 cells) (both from Dharmacon), or HiPerfect (RPE cells) (Qiagen) according to manufacturer’s protocol. siRNAs were transfected in a pool consisting of two distinct siRNAs in all experiments except where noted.

Immunoblots and Immunoprecipitation

Whole cell lysates were obtained using non-denaturing lysis buffer (NDLB) (50 mM Tris-Cl (pH 7.4), 300 mM NaCl, 5 mM EDTA, 1% (w/v) Triton X-100, 1 mM PMSF, 1 mM sodium orthovandate, 10 mM NaF, and phosphatase inhibitor cocktail (Roche)). Soluble fractions were prepared for immunoblotting. Cytoplasmic fractionation was performed as previously described (Thorne et al., 2010). Cells were incubated on ice in lysis buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovandate, 10 mM NaF, and phosphatase inhibitor cocktail (Roche)), scraped, transferred to microfuge tubes, and NP-40 was added to 0.5%. Lysates were vortexed and sheared 8 times using a 23-gauge needle. Lysates were centrifuged at 16,000×g for 2 min. Supernatants (cytoplasmic fractions) were recovered. For co-immunoprecipitations, cell lysates were prepared using NDLB. Lysates were diluted to 1 mg/mL and incubated with antibody for 1 h in a TOMY shaker at 4°C followed by addition of Protein G magnetic beads (New England Biolabs) for 1 h. Beads were washed 3 times with 10× volumes of NDLB. Proteins were eluted from beads with protein sample buffer and analyzed by SDS-PAGE and immunoblotting. Chemiluminescence signal was detected using a C-DiGit blot scanner (LI-COR).

Generation of the mAb7E5 Antibody

A cDNA fragment corresponding to the extracellular domain of human LRP6 (amino acids 589–1244) was cloned into the pMAL vector (NEB) and transformed into BL21(DE3) cells (Invitrogen). Briefly, cells were grown in LB and induced with 300 µM IPTG for 4 h at room temperature. Cells were then centrifuged at 2,500 RPM for 30 min. Pellets were re-suspended in Buffer A (50 mM Tris pH 8.0, 200 mM NaCl, 0.1% Tween-20 and 1 mM PMSF), sonicated, and centrifuged at 12,000 RPM for 30 min. The supernatant was then incubated with amylose resin (NEB) for 2 h with rocking at 4°C. Resin was washed 2× with Buffer A, 1× with Buffer B (50 mM Tris pH 8.0, 50 mM NaCl, and 1 mM PMSF), and bound proteins eluted with 1% maltose in Buffer B. The purified protein was used to inoculate BALB/c mice for monoclonal antibody production by the Vanderbilt Protein and Antibody Core. Fusion clones (1,500) were initially screened based on their capacity to bind the antigen as assessed by ELISA. Of these clones, eight were found to inhibit Wnt3a-stimulated signaling in HEK293 STF cells but not endogenous Wnt signaling in the colorectal cancer cell line, SW480. Four additional clones were found to inhibit Wnt3a-induced signaling in HEK293 STF cells as well as endogenous Wnt signaling in SW480 cells. Of these four clones, mAb7E5 was found to be the most potent in both cell lines and was chosen for further characterization.

Generation of RKO APC^KO

Two APC gRNA sequences (5’-CCCCCTATGTACGCCTCCC-3’ and 5’-CTTTGACAAACTTGACTTT-3’) were cloned into pCAS-Guide-eGFP (Origene) using BamHI and BsmBI. APC gRNA oligos were designed with a 5’-GATCG overhang and extra guanidine nucleotide on the 3’-end (Sigma). Oligonucleotides were annealed as per manufacturer’s protocol using a 4-step PCR procedure. Digested pCAS-Guide-eGFP vector was ligated to the annealed oligos using T4 ligase. Final clones were sequence-verified. Plasmids containing the two APC gRNA sequences were transfected into RKO cells at 80% confluence using Fugene HD. Transfected cells were sorted by flow cytometry (Vanderbilt Flow Cytometry Core) as single cells into a 96-well plate. Single colonies were grown to confluence and split into 24-well plates. Clones were propagated and screened by TOPflash. Clones showing activation compared to vector control were screened by immunofluorescence and stained for β-catenin. Clones with high β-catenin levels were further screened by immunoblotting.

Generation of Stable Cell Lines

To obtain lentivirus, 293FT cells were seeded at 1.2×10⁶ on 60mm dish in 3 mL of DMEM, 10% FBS, without antibiotic. The next day, cells were transfected with 6.6µg lentiviral DNA contruct (lentiviral TOPflash, pLKO.1 APCshRNA, or pBabe TIR1-9myc), with 5µg psPAX2 (or puMVC for pBabe 9myc-TIR1), 2µg pMD2.G, 21µl 2MCaCl₂, and sterile water (for a final volume of 170µL), mixed with 170µΛ 2× HBS by bubbling air through the mixture. Cells were incubated with 25mM Chloroquine for 0min before adding the DNA mix. After 5–7 h, the transfection media was replaced with fresh DMEM+10% FBS, and media containing lentivirus or retrovirus was harvested after 72 h.

RKO, DLD1, HCT116 WT KO, and RPE cells were incubated with 1.5 mL of TOPflash or APC shRNA lentivirus, and RKO APC^KO cells were incubated with 1.5 mL 9myc-TIR1 retrovirus with 12 µg polybrene (hexadimethrine bromide) for 4 h. Media was removed and cells incubated for 48 h prior to selection with puromycin or G418. To generate MEF LRP6^KO, primary MEF LRP6^flox cells were immortalized by transfecting SV40 and selected with 500µg G418 for 1 week. Cells were then transfected with pICG mCherry-IRES-Cre, and mCherry positive cells (MEF LRP6^KO) were isolated by FACS.

Sucrose Density Gradients

Lysates from RKO, RKO incubated for 2 h with 20 ng recombinant Wnt3a (Times Bioscience), and RKO APC^KO were prepared using NDLB as described in the Immunoblots and immunoprecipitation section. Lysates and MW standards (BioRad) were layered on top of sucrose gradients containing equal volumes of 5%, 10%, 20%, and 30% sucrose in PBS (137 mM NaCl, 7 mM Na₂HPO₄, and 3 mM NaH₂PO₄, pH 7.2). Gradients were centrifuged for 4 h at 46,000 rpm in a SW55Ti swinging-bucket rotor (Beckman Coulter). Fractions were collected, precipitated using a protein precipitation kit (National Diagnostics) according to manufacturer’s protocol, resuspended in 6× Sample Buffer, and analyzed by immunoblotting.

Endocytosis Assays

Cells were plated in four 35 mm dishes and incubated at 37°C with L-cell conditioned media (L CM), Wnt3a Conditioned Media (Wnt3a CM) or 20 ng/mL recombinant Wnt3a (Time Bioscience), 30 mM LiCl, or 2 µM CHIR-99021 for 24 h. Cells were then incubated at 4°C for 3 h. Cells were lysed in NDLB at 0, 1, 2, and 3 h post temperature shift. For recovery assays, dishes were then shifted back to 37°C, and lysates were taken at 0, 1, 2, and 3 h. For small molecule experiments, cells were preincubated for 30 min with chloroquine, pitstop-2, or nystatin as indicated, incubated with 20 ng/mL recombinant Wnt3a (Time Bioscience) for 2 h, and either analyzed by confocal microscopy or washed with ice cold PBS and lysed with NDLB.

Microscopy

For β-catenin staining, cells were fixed in methanol at −20°C, washed in TBS plus 0.1% Triton X-100, blocked in Abdil buffer (TBS, 0.1% Triton X-100, 2% BSA, and 0.1% sodium azide) overnight at 4°C, washed with TBS plus 0.1% Triton X-100, and incubated with anti-β-catenin antibody (1:500, Vanderbilt Protein and Antibody Resource). Cells were incubated with Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:1000) and mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen). Images were acquired using a CoolSNAP ES camera mounted on a Nikon Eclipse 80i fluorescence microscope with 40× and 60× objectives. For immunostaining of APC, clathrin, and caveolin-1, RPE cells were seeded on 12 mm glass coverslips, cultured for 24 h, left untreated or treated with Wnt3a ligand (20 ng/mL), incubated in tissue culture media for an additional 2 h, fixed at 37°C for 20 min in 4% paraformaldehyde in PBS, rinsed 3× with 100 mM glycine in PBS, and incubated in 0.1% Triton X-100 in PBS with 5% glycine and 5% goat serum for 1 h at room temperature. Cells were incubated with mouse anti-APC antibody (Santa Cruz) plus rabbit anti-clathrin heavy chain monoclonal antibody (1:200, Cell Signaling) or rabbit anti-caveolin-1 polyclonal antibody (1:200, BD Transduction Laboratories) in blocking buffer with 0.05% Tx-100 for 2 h at room temperature, rinsed with PBS, incubated for 1 h in Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies (1:300), and washed in PBS. Coverslips were mounted on glass slides with Prolong Gold Antifade Reagent. For Golgi immunostaining, RKO APC^KO TIR1 cells were incubated with Brefeldin A (5µg; Sigma) or DMSO as indicated and fixed as described above. Cells were immunostained using anti-Giantin (1:200, Abcam) and DRAQ5 (Thermo Fisher). Fluorescent images were acquired using a Zeiss LSM 510 confocal microscope (Carl Zeiss Microscopy, Inc.; Thornwood, NY) with a Plan-Apochromat 100×/1.4 oil Neofluar oil immersion objective and Argon/2 30 mW laser (458, 488, 514 nm) or HeNe lasers (543 or 633 nm).

Live Cell Imaging

Cells were plated in 35 mm glass bottom dishes (MatTek Corporation) at ~50% confluency and transfected with LRP6-eYFP for 6 h. Media was replaced by red phenol-free DMEM + 10% FBS. Cells were pre-incubated for 30 min with small molecules or mAb7E5 and/or incubated with 20 ng recombinant Wnt3a for 2 h. For time-lapse live cell imaging one control stack was taken of 7–10 cells using “mark and find” (Leica Application Suite). Wnt3a was added to the MatTek dish, refocusing was done, and subsequent z-stacks of the same cells were taken every 9 min for 3 h. Imaging was performed on a Leica SP5 confocal microscope using the Live Cell Controller by Live Cell at 37°C and 85–90% relative humidity. The 561 nm laser was used for excitation, and the laser power remained the same during acquisition for each experiment. This eliminated cells not visible under a certain threshold of brightness to account for heterogeneity in plasmid expression of transiently transfected plasmids.

Organoid Lentiviral Transduction

Lentivirus was packaged in HEK293T cells using pMD2.G and psPAX2 vectors and either the GIPZ Non-silencing Lentiviral shRNA control vector (Dharmacon, RHS4346) or two pooled GIPZ shLRP6 vectors (V3LMM_484540 and V3LMM_484536). 30 mL of lentiviral supernatant was harvested, filtered through a 0.45 mm filter, and concentrated overnight via centrifugation at 8,000×g. The lentiviral pellet was resuspended in 250 µL L-WRN media with 8 mg/mL polybrene and 10 mM Y27632. APC^min organoids were collected and Matrigel-digested for 30 min on ice using Cell Recovery Solution (Corning). Organoids were pelleted at 150×g for 5 minutes then resuspended in 1 mL cold PBS and sheared through a 25G needle 1×. Dissociated organoids were pelleted, resuspended in 250 mL concentrated lentivirus, spinoculated at 600 × g for 1 h at 32°C, incubated at 37°C for an additional 6 h, pelleted, resuspended in Matrigel, plated in 12-well plates, and overlayed with 0.5 mL basal media for 4 days.

Organoid Immunofluorescence

APC^min organoids were fixed in 4% formaldehyde in PBS for 30 min to 1 h at room temperature in 1.5 mL tubes, permeabilized with 0.5% Triton X-100 in TBS for 30 min at room temperature, incubated overnight at 4°C in blocking buffer (PBS, 1% BSA, 3% normal goat serum, 0.2% Triton X-100), followed by overnight incubation at 4°C in blocking buffer with mouse anti-β-catenin antibody (1:500, Vanderbilt Protein and Antibody Resource). Organoids were then washed 3× for at least 30 min in 0.1% Triton X-100 in TBS and incubated 2–3 h at room temperature in blocking buffer with Cy3-conjugated anti-mouse secondary antibody (1:500, Jackson Immuno). Stained organoids were incubated with Hoescht (1:1000) in TBS for 15 min at room temperature and mounted in Mattek dishes with ProLong Gold Antifade Reagent (Invitrogen). Imaging was done using a Nikon Spinning Disk microscope with Andor DU-897 EMCCD camera and 561 nm and 405 nm lasers.

Drosophila RNAi Experiments

Temporal RNAi-mediated knockdown in progenitor cells was performed using the temperature-sensitive progenitor cell driver esg^ts. For concomitant Apc1 and arr knockdown, crosses were maintained at 18°C until late third instar larval stage then shifted to 29°C restrictive temperature to allow RNAi-mediated knockdown during pupation. For concomitant Apc1 and dsh knockdown, crosses were maintained at 18°C until second instar larval stage then shifted to 29°C.

To induce knockdown of arr, dsh, wg, and wls in an Apc1^Q8 background, crosses were maintained at 18°C until late third instar larval stage then shifted to 29°C restrictive temperature. Expression of the Wg target gene reporter nkd-lacZ was analyzed in Apc1^Q8 mutants with and without expression of arr and dsh RNAi.

Genotype for arrow knockdown in Apc1 null mutant (Figure 8): esg-Gal4 tubGal80^ts UAS-GFP/ UAS-arrow RNAi; Apc1^Q8

Genotype for dsh knockdown in Apc1 null mutant (Figure 8): esg-Gal4 tubGal80^ts UAS-GFP/ UAS-dsh RNAi; Apc1^Q8

Genotype for simultaneous knockdown of arrow and Apc1 (Figure S7): UAS-dicer2/+; esg-Gal4 tubGal80^ts UAS-GFP/+ or UAS-arrow RNAi; UAS-Apc1 RNAi/UAS-arrow RNAi or +.

Genotype for simultaneous knockdown of dsh and Apc1 (Figure S7): UAS-dicer2/+; esg-Gal4 tubGal80^ts UAS-GFP/+ or UAS-dsh RNAi; UAS-Apc1 RNAi/UAS-dsh RNAi or +.

Genotype for arrow knockdown in Apc1 null mutant expressing nkd-lacZ (Figure S8): esg-Gal4 tubGal80^ts UAS-GFP/ UAS-arrow RNAi; Apc1^Q8/ nkd-lacZ Apc1^Q8.

Genotype for dsh knockdown in Apc1 null mutant expressing nkd-lacZ (Figure S8): esg-Gal4 tubGal80^ts UAS-GFP/ UAS-dsh RNAi; Apc1^Q8/ nkd-lacZ Apc1^Q8.

Genotype for yellow knockdown in Apc1 null mutant expressing nkd-lacZ (Figure S8): esg-Gal4 tubGal80^ts UAS-GFP/ UAS-y RNAi; Apc1^Q8/ nkd-lacZ Apc1^Q8.

Genotype for wg knockdown in Apc1 null mutant (Figure 8): esg-Gal4 tubGal80^ts UAS-GFP/ UAS-wg RNAi; Apc1^Q8

Genotype for wls knockdown in Apc1 null mutant (Figure 8): esg-Gal4 tubGal80^ts UAS-GFP/ UAS-wls RNAi; Apc1^Q8

Drosophila Immunohistochemistry

Adult intestines (2 d) were dissected in PBS, fixed in 4% paraformaldehyde in PBS for 45 min, washed in PBS plus 0.1% Triton X-100, incubated in PBS plus 0.1% Tween-20 and 10% BSA for 1 h at room temperature, and incubated at 4°C overnight in PBS plus 0.5% Triton X-100 with chicken anti-GFP antibody (1:10000, Abcam), mouse anti-β-gal (Promega, 1:500), mouse anti-Arm (Developmental Studies Hybridoma Bank, [DSHB], 1:20), and mouse anti-Prospero (DSHB, 1:100). Samples were stained with secondary antibody (goat anti-chicken Alexa Fluor 488 and anti-mouse Alexa Fluor 555 conjugates; ThermoFisher Scientific, 1:500) for 2 h at room temperature, stained with DAPI (2 µg/mL), and mounted in Prolong Gold (Invitrogen). Confocal images were obtained from the R4 region of the posterior midgut with a Nikon A1RSi confocal microscope with 60× lens, and the number of esg-positive cells in a field of 0.051 mm² was counted (>15 posterior midguts analyzed per genotype). Images were processed using Adobe Photoshop software. The intensity of nkd-lacZ expression along a line drawn across the posterior midgut was quantified using NIS-Elements software (Nikon) from images obtained with a 20× lens. The distance between the start of nkd-lacZ expression at the hindgut/midgut boundary and the last peak of high intensity nkd-lacZ expression in the posterior midgut was measured.

Auxin-Dependent Degradation Assay

RKO APC^KO TIR1 cells were plated in 35 mm dishes and transfected with pCS2 Myc-APC^T or pCS2 AID-Myc-mCherry-APC^T. At 24 h post-transfection, cells were incubated with 500 µM auxin analog 1-Naphthaleneacetic acid (Sigma). For Brefeldin A experiments, cells were pre-incubated with 5 µg Brefeldin A or DMSO before addition of auxin. Timepoints were taken every 30 min for 2 h. Cells were lysed in NDLB and immunoblotted.

Reporter Assay

For cell-based luciferase assays, HEK293 STF, RKO STF, SW480 STF, DLD1 STF, HCT116 STF, HCT116 WT KO STF, and HEK293 CMV-Luc cells were seeded into 24-well plates at ~50% confluency. Cells were incubated with L CM, Wnt3a CM, 20 ng/mL purified Wnt3a (Time Bioscience), or 30 mM LiCl prior to lysis with 1× Passive Lysis buffer (Promega). Luciferase expression was measured using Steady-Glo Luciferase Assay (Promega). For inhibitor studies, cells were incubated with mAb7E5, Wnt-C59 (Cellagen Technology), IWP-2 (StemRD) or Fz8-Fc (R&D Systems) for 24 h (or 48 h for SW480, DLD1, and HCT116 cells) prior to lysis. Luciferase signal was normalized to viable cell number using CellTiter-Glo Assay (Promega). Alternatively, TOPflash experiments in HEK293, HEK293 Dvl TKO, MEF, MEF PORCN^KO, MEF Caveolin-1^KO, RKO, and RKO APC^KO cells were performed as follows. Cells were plated, co-transfected with TOPflash and Renilla expression plasmids at 24 h, and lysed using Dual-Glo Assay (Promega) 24 h later. Luciferase signal was normalized to co-transfected Renilla expression. Data were normalized to the signal obtained from positive controls (plotted as 100% activation). Assays were performed in quadruplicate and repeated at least 3×.

RNA Isolation and qRT-PCR

HEK293 cells were treated with LRP6 siRNA and APC siRNA for 72 h, harvested using STAT60 reagent (Amsbio) according to manufacturer’s protocol, and further purified using RNeasy purification kit (Qiagen). RNA (2 µg) was reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific). qRT-PCR was performed on a Bio-Rad C1000 thermocycler using predesigned and revalidated TaqMan probes that span exons (Thermo Scientific). GAPDH was used as reference control. Technical duplicates were performed for each gene.

Immunoblotting Quantification

Chemiluminescence signal from immunoblots were detected using a C-DiGit blot scanner (LI-COR). Obtained images and band intensity were analyzed using Image Studio (LI-COR).

Line Scan Analysis

Images or maximum-intensity projections of z-stacks were analyzed using ImageJ/FIJI (National Institutes of Health, Bethesda, MD). Line scan analyses to quantify nuclear β-catenin or LRP6-eYFP signal intensity were performed using ImageJ. At least five representative cells were measured per condition.

Organoid Measurement

To measure organoid size, a straight line was drawn across the diameter of APC^min organoids. The number of pixels were quantified and normalized to untreated control. At least 20 organoids per condition were analyzed. All measurements were performed using Image J/FIJI (National Institutes of Health, Bethesda, MD).

Statistical Analysis

All graphs were generated using Prism (GraphPad Software, Inc.). Statistical analyses were performed using a two-tailed, unpaired Student’s t-test, except for colocalization analyses. A value of p<0.05 is considered statistically significant.

Colocalization Analysis

For colocalization analysis, Intensity Correlation Analysis plugin in FIJI was used. Pearson’s correlation coefficients were calculated using 4× zoom images. For each condition, 10–19 images were analyzed. Box and whisker plots were used for reporting Pearson’s correlation coefficients. A two-tailed, unpaired, non-parametric Mann-Whitney U-test was used for calculating p-values.