Cd184

CAF, MCF-10A, ZR-75-1, HCT116, QBC939, NCCIT, RBE, HUCCT1, ZJU-0826, and ZJU-1125 cells were washed thrice with ice-cold phosphate buffered saline (PBS), lysed in RIPA buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% sodium deoxyholate, 1% Nonide P-40, and 0.1% SDS with Pierce Protease Inhibitor Tablets (AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstain A) (Thermo Fisher Scientific Inc., Waltham, MA, USA) and separated by centrifugation. The protein concentration was detected using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc). Antibodies against the following proteins were used: E-cadherin, N-cadherin, β-catenin, α-SMA, MUC1, CD146, SOX17, Vitamin D3 Receptor (VDR), pdx1, CD326, FoxA1/HNF3α, FoxA2/HNF3β, Nanog, GAPDH, and β-actin (all 1:1000, from Cell Signaling Technology, Danvers, MA, USA).
Flow cytometry was performed using a FACScan instrument (BD Bioscience, San Jose, CA, USA) and commercially available reagents for cells at passages 3–5 and 60–65. A panel of monoclonal antibodies was evaluated, including CD24, CD44, CD29, CD34, CD90, CD117, CD133, CD184, CD326, and CD338 (Biolegend, San Diego, CA, USA). Antigen expression was determined based on a significant shift in staining compared to an isotype control.

The expression of CD90, CD49f, CD93 and CD184 (CXCR4) on the surface of 18Lin⁻CD34⁺CD38⁻CD133⁺GPI-80⁺ and 18Lin⁻CD34⁻CD133⁺GPI-80⁺ cells was analyzed using FACSCantoII (BD Biosciences). Human CB-derived Lin⁻ cells obtained from individual samples (n = 7) were stained with a mixture of FITC-conjugated 18 Lin mAbs, PE-conjugated anti-GPI-80 mAbs, PE-Cy7-conjugated anti-CD38 mAb, APC-conjugated anti-CD133/1 mAb, APC-Cy7-conjugated anti-CD34 mAb (BioLegend), BV510-conjugated anti-CD45 mAb and biotin-conjugated anti-CD90 (BioLegend), CD49f (BioLegend), CD184 (BioLegend) or CD93 mAb (OriGene). Biotin-conjugated mAbs were then reacted with BV421-conjugated streptavidin for 30 min at 4 °C. The samples were washed twice with PBS⁻/FCS followed by 7-AAD staining before the analysis. The positive gate for the BV421 fluorescence channel was defined according to the FMO control.