D on the criterion of having at least one MS/MS data with Mascot scores that exceeded the thresholds (p,0.01).RNA InterferenceThe shRNA oligonucleotide sequences were as follows: CD133#1 (59-GGAUACACCCUACUUACUAAA-39), CD133#2 (59-GCACUCUAUACCAAAGCGUCA-39), desmoglein-2#1 (59GUUAGCGAGAGCAUGGAUAGA-39), desmoglein-2#2 (59GCAUUAUCAGUAAUAAUUUGU-39), plakoglobin (59-GAGCAUGAUUCCCAUCAAUGA-39).Lentivirus ProductionLentiviral vector (CS-RfA-CG) expressing an shRNA driven by the H1 promoter was transfected with the packaging vectors pCAG-HIV-gp and pCMV-VSV-G-RSV-Rev into 293FT cells using Lipofectamine 2000 Transfection Reagent (Life Technologies). All plasmids were kindly provided by H. Miyoshi (RIKEN BioResource Center, Japan). Viral supernatant was purified by ultracentrifugation at 25,000 rpm for 90 min (SW28 rotor, Beckman). Infection efficiency was monitored by green fluorescence protein (GFP) expression as it is driven by the CMV promoter.AntibodiesMouse monoclonal antibody (mAb) to CD133 (AC133) was 18325633 obtained from Miltenyi Biotec. Mouse mAb to desmocollin-2/3 (7G6), desmoglein-2 (6D8) and a-tubulin were from Santa Cruz Biotechnology. Mouse mAb to plakoglobin was from BD Biosciences. Mouse mAb to GAPDH was from Millipore. Mouse mAb to desmoplakin (2Q400) was from Abcam and used for immunohistochemistry. Rabbit polyclonal antibody (pAb) to desmoplakin was from Abcam and used for immunoblotting.ImmunohistochemistryCells were plated onto laminin-coated glass coverslips and fixed with ice-cold methanol. Cells were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with Alexa 488 or 594 (Life Technologies). For CD133 visualization, cells were incubated with anti-CD133 antibody conjugated to R-phycoerythrin. Lecirelin TO-PRO-3 iodide (Life Technologies) was used for nuclear DNA staining. Cells were photographed with an LSM510 META laser scanning microscope (Carl Zeiss).Immunoprecipitation and ImmunoblottingThe membrane fraction of cells was lysed in lysis buffer [50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 NP40, 1 mM dithiothreitol and protease inhibitor cocktail (Roche)] (Fig. 1D). Alternatively, the membrane fraction was lysed in lysis buffer JSI124 containing 1 digitonin instead of NP40 (Fig. 1F). Lysates were incubated with anti-CD133, anti-plakoglobin or control IgG1 immobilized to Activated CH-Sepharose 4B (GE Healthcare) for 4 h at 4uC. After five washes with lysis buffer, bound proteins were eluted with 100 mM Glycine-HCl and subjected to trypsin digestion. After desalted with ZipTip (C18; Millipore), the sample was subjected to liquid chromatography-mass spectrometry. For immunoblotting, the eluted proteins were fractionated by SDSPAGE and transferred to a PVDF membrane (Immobilon-P, Millipore). The membrane was subjected to immunoblot analysis using alkaline phosphatase-conjugated anti-mouse or rabbit IgG (Promega) as secondary antibodies. Visualization was performed using the NBT/BCIP colorimetric substrate system (Promega).Cell Dissociation Assay56104 cells were seeded in laminin-coated 12-well plates. Cells were detached from culture plates with a cell scraper and passed 10 times through a 200-ml pipette tip in PBS containing 1 mM CaCl2 and 0.5 mM MgCl2. The images were captured by phase contrast microscopy. The extent of cell dissociation was represented by the ratio of cell number/cluster number.Hanging Drop Cell Aggregation AssayCells were trypsinized, washed with PBS and resuspended at 56105 cells per mil.D on the criterion of having at least one MS/MS data with Mascot scores that exceeded the thresholds (p,0.01).RNA InterferenceThe shRNA oligonucleotide sequences were as follows: CD133#1 (59-GGAUACACCCUACUUACUAAA-39), CD133#2 (59-GCACUCUAUACCAAAGCGUCA-39), desmoglein-2#1 (59GUUAGCGAGAGCAUGGAUAGA-39), desmoglein-2#2 (59GCAUUAUCAGUAAUAAUUUGU-39), plakoglobin (59-GAGCAUGAUUCCCAUCAAUGA-39).Lentivirus ProductionLentiviral vector (CS-RfA-CG) expressing an shRNA driven by the H1 promoter was transfected with the packaging vectors pCAG-HIV-gp and pCMV-VSV-G-RSV-Rev into 293FT cells using Lipofectamine 2000 Transfection Reagent (Life Technologies). All plasmids were kindly provided by H. Miyoshi (RIKEN BioResource Center, Japan). Viral supernatant was purified by ultracentrifugation at 25,000 rpm for 90 min (SW28 rotor, Beckman). Infection efficiency was monitored by green fluorescence protein (GFP) expression as it is driven by the CMV promoter.AntibodiesMouse monoclonal antibody (mAb) to CD133 (AC133) was 18325633 obtained from Miltenyi Biotec. Mouse mAb to desmocollin-2/3 (7G6), desmoglein-2 (6D8) and a-tubulin were from Santa Cruz Biotechnology. Mouse mAb to plakoglobin was from BD Biosciences. Mouse mAb to GAPDH was from Millipore. Mouse mAb to desmoplakin (2Q400) was from Abcam and used for immunohistochemistry. Rabbit polyclonal antibody (pAb) to desmoplakin was from Abcam and used for immunoblotting.ImmunohistochemistryCells were plated onto laminin-coated glass coverslips and fixed with ice-cold methanol. Cells were incubated with primary antibodies followed by incubation with secondary antibodies conjugated with Alexa 488 or 594 (Life Technologies). For CD133 visualization, cells were incubated with anti-CD133 antibody conjugated to R-phycoerythrin. TO-PRO-3 iodide (Life Technologies) was used for nuclear DNA staining. Cells were photographed with an LSM510 META laser scanning microscope (Carl Zeiss).Immunoprecipitation and ImmunoblottingThe membrane fraction of cells was lysed in lysis buffer [50 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 NP40, 1 mM dithiothreitol and protease inhibitor cocktail (Roche)] (Fig. 1D). Alternatively, the membrane fraction was lysed in lysis buffer containing 1 digitonin instead of NP40 (Fig. 1F). Lysates were incubated with anti-CD133, anti-plakoglobin or control IgG1 immobilized to Activated CH-Sepharose 4B (GE Healthcare) for 4 h at 4uC. After five washes with lysis buffer, bound proteins were eluted with 100 mM Glycine-HCl and subjected to trypsin digestion. After desalted with ZipTip (C18; Millipore), the sample was subjected to liquid chromatography-mass spectrometry. For immunoblotting, the eluted proteins were fractionated by SDSPAGE and transferred to a PVDF membrane (Immobilon-P, Millipore). The membrane was subjected to immunoblot analysis using alkaline phosphatase-conjugated anti-mouse or rabbit IgG (Promega) as secondary antibodies. Visualization was performed using the NBT/BCIP colorimetric substrate system (Promega).Cell Dissociation Assay56104 cells were seeded in laminin-coated 12-well plates. Cells were detached from culture plates with a cell scraper and passed 10 times through a 200-ml pipette tip in PBS containing 1 mM CaCl2 and 0.5 mM MgCl2. The images were captured by phase contrast microscopy. The extent of cell dissociation was represented by the ratio of cell number/cluster number.Hanging Drop Cell Aggregation AssayCells were trypsinized, washed with PBS and resuspended at 56105 cells per mil.
