C2206

Cells were cultured on glass coverslips in 6-well plates to at least 80% confluence and fixed in ice-cold methanol for 20 min for both proximity ligation assays (PLA) E-cadherin/b-catenin and E-cadherin/p120. PLA was performed using Duolink Detection kit (Olink Bioscience, Sweden), according to the manufacturer’s instructions for Duolink Blocking solution and Detection protocol. Briefly, slides were blocked, incubated with antibodies directed against E-cadherin cytoplasmic domain (610,182, BD Biosciences or Clone 24E10, #3195, Cell Signaling, USA), b-catenin (C2206, Sigma-Aldrich, USA) and p120 (610,134, BD Biosciences, USA), followed by incubation with the secondary PLA probes (anti-mouse Minus and anti-rabbit Plus) conjugated to unique oligonucleotides. Amplification template oligonucleotides were hybridized to pairs of PLA probe and circularized by ligation. Rolling circle amplification was performed and detection of amplified DNA was possible by addition of complementary oligonucleotides labeled with Cy3 fluorophore. Coverslips were mounted on Vectashield with DAPI (Vector Laboratories, USA). Images were acquired on a Carl Zeiss Apotome Axiovert 200 M Fluorescence Microscope (× 20 and × 40 objectives; Carl Zeiss, Germany) with an Axiocam HRm camera and processed with the Zeiss Axion Vision 4.8 software. Quantification of PLA signals was achieved using BlobFinder V3.2.42.

The primary antibodies are listed in Table S1. They were used at the following dilutions for either immunoblotting or IF: rabbit anti-GFP (A-11122, Thermo Fisher Scientific, IB: 1/2000, IF: 1/200); mouse GFP (11814460001, Roche, IF: 1/100); mouse myc (9E10 (purified), in-house, IF: 1/100); mouse HA (32-6700, Thermo Fisher Scientific, IB: 1/1000); rabbit ß-catenin (C2206, Sigma, IB: 1/3500, IF: 1/500); goat VE-cadherin (sc-6458, Santa Cruz, IF: 1/1000); rat anti-ZO-1 (R40.76, a kind gift from Daniel Goodenough, Harvard Medical School, IF: 1:100); mouse anti-E-cadherin (610181, BD Biosciences, IF: 1/2500); mouse anti-β-tubulin (32-2600, Thermo Fisher Scientific, IB: 1/3500); mouse anti-PanPMCA (MA-3914 [clone 5F10], Thermo Fisher Scientific, IF: 1/1000, IB: 1/1000); rabbit anti-PLEKHA7 (Rb30388, in-house (2 ), IF: 1/1000); guinea pig anti-PLEKHA7 (GP2737, in-house (3 (link)), IF: 1/200); rabbit anti-PDZD11 (Rb29958 (3 (link)), IF: 1/50 to 1/100); and rat anti-Crb3a (1E6, a kind gift from André Le Bivic [IBDM, University of Marseille], IB: 1/1000).
Secondary antibodies (Table S1) for IF from Jackson ImmunoResearch were diluted at 1/300 and from Invitrogen at 1/100. Horseradish peroxidase–conjugated secondary antibodies used for IB (antimouse, antirat, and anti-rabbit were diluted 1/20000).

Brains and eyes were harvested from Stages 40/41 embryos that were anesthetized with 0.4mg/ml MS222 in 1X MBS. The tissues were mechanically homogenized with radioimmunoprecipitation assay (RIPA; Sigma) buffer supplemented with protease inhibitor cocktail (Roche) by repeated pipetting, and solubilized by constant rotation at 4°C for 30 min. Remaining non-solubilized material was then pelleted by centrifugation at 13000rpm for 10 min and only the supernatant was then retained. Protein concentration was determined by Bradford assay (Bio-Rad) and spectrophotometry. Bovine serum albumin (BSA; Invitrogen) was used to create a standard curve for protein concentration and for normalizing the concentration between samples. The lysates were resolved by 12% TGX precast gels (BioRad) at constant 20mA, transferred to nitrocellulose membrane (BioRad) at constant 110V and subjected to western blot analysis by incubating with a rabbit anti-β-actin (Abcam Cat# ab8227, RRID:AB_2305186; 1:8000) or rabbit anti-β-catenin (Sigma-Aldrich Cat# C2206, RRID:AB_476831; 1:8000) antibody at 4°C for overnight. The blots were then incubated with HRP-conjugated secondary antibodies (Abcam Cat# ab97080, RRID:AB_10679808; 1:16000) in room temperature for one hour, followed by ECL-based detection (Invitrogen).

Colorimetric in situ hybridizations were performed as described previously (Rentzsch et al., 2006 (link), 2008 (link)). Fixation for in situ hybridization, immunostaining (anti-acetylated tubulin antibody, Sigma T6793, batch 081M4760, 1:500; anti-mouse β-catenin, Sigma C2206, batch 062M4806, 1:500), Phalloidin and DAPI staining were performed as described by Leclere and Rentzsch (2014 (link)).

Proteins were extracted on ice using a RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor‐cocktail (Roche). SDS‐PAGE and western bolt were performed to analyse the expression of target proteins according to standard protocols. Primary antibodies in western blotting examination included the following: myogenin (ab1835">ab1835, Abcam), MHC (ab180779">ab180779, Abcam), GAPDH (D16H11, CST), ITGB1 (ab179471">ab179471, Abcam), ITGB1D (97733, NOVUS), ITGB1D (ab8991, Abcam), LRP6 (C5C7, CST), α‐Tubulin (1124‐1‐AP, Proteintech), Histone H3 (ab1791">ab1791, Abcam), GFP (2555, CST), hnRNP A1 (ab123378, Abcam), PTBP1 (ab133734">ab133734, Abcam), β‐catenin (C2206, Sigma). The secondary antibodies conjugated to infrared dyes (LI‐COR Biosciences) were applied at a concentration of 1:10 000, and the blots were visualized using an Odyssey imager (LI‐COR Biosciences).

Total mRNA was reverse‐transcribed into cDNA (AT301 TransGen Biotech, Beijing, China), and real‐time quantitative PCR was performed with CFX96 Real‐Time PCR Detection System (Bio‐Rid, Hercules, CA, USA). For western blot, the protein from cell extracts was separated by 10% SDS‐PAGE electrophoresis and was later transferred onto PVDF membrane. Membranes were incubated with ESR1 (1:3000, EPR4097, ab108398; Santa Cruz, Dallas, TX, USA), TCF4 (1:2000, ab217668; Abcam, Cambridge, MA, USA), CMYC (1:2000, ab32072; Abcam), LEF1 (1:2000, ab137872; Abcam), WNT1 (1:1000, ab15251; Abcam), CTNNB1 (1:1000, C2206; Sigma‐Aldrich, St. Louis, MO, USA), VINCULIN (1:5000, #18799; Cell Signaling Technology, Danvers, MA, USA), and then detected using ECL Blotting Detection Reagents (Merck Millipore, Burlington, MA, USA). Cells of different groups were suspended in DMEM/F12 Medium supplemented with 20 ng/mL EGF (BD Biosciences, San Jose, CA, USA), bFGF and 4 μg/mL insulin (Sigma), and then plated in 6‐well ultra‐low attachment dishes (1000 cells/mL; Corning Incorporated, Tewksbury, MA, USA). To analyse the self‐renewal ability, sphere number of each captured image was counted by using phase contrast microscope (Nikon, Tokyo, Japan).

PMCs were grown on gelatin-treated coverslips and fixed with PFA (4%) for 15 min. After permeabilization in 1% Tween-20 and blocking, cells were incubated for 1 hr with rabbit anti–β-catenin (SIGMA, C2206) and mouse monoclonal anti-Snail1 (8). After washing, samples were incubated with secondary antibodies anti-murine IgGs (labelled with Alexa 555) or anti-rabbit IgGs (labelled with Alexa 647 (1/500) (all from Invitrogen). Cells were mounted with Flouromount G–DAPI to counterstain nuclei and visualized in a Leica SPE confocal microscope.
Matrigel-released cultured cells were fixed with PFA (4%) and extended on a coverslip after a quick spin (30 sec, 400 × g). They were fixed again with PFA for 5 min, washed, permeabilized in 0.3% Triton X-100, and incubated with antibodies against E-cadherin (Transduction Labs), amylase (Sigma-Aldrich), or CK19 (Abcam). After washing, samples were incubated with secondary antibodies labelled with Alexa 488, Alexa 555, or Alexa 647 (Invitrogen) and analysed in the confocal microscope.