E-cadherin binds and stabilizes β-catenin in human EBs. (A) CDH1 (encoding E-cadherin) and CTNNB1 (encoding β-catenin) expression in hematopoietic and stromal cell lineages, derived from a publicly available single-cell RNA-sequencing database.²⁸ (B) Representative flow cytometry plot, used to define erythroid differentiation stages based on CD71 and CD235a expression, of cultured human EBs derived from CD34⁺ mobilized HSPCs, revealing both (C) E-cadherin and (D) β-catenin protein expression to increase upon EB formation. (E-F) Western blot analysis of the association of E-cadherin with β-catenin in cultured human EB by immunoprecipitation of endogenous E-cadherin and β-catenin, using isotype immunoglobulin G (IgG) antibody and empty beads as negative controls, as also defined by western blot analysis of the flow through. (G) Representative immunofluorescent staining of cultured EBs on VCAM-1–coated slides, revealing E-cadherin (red) and β-catenin (green) to colocalize at the plasma membrane and to be absent in the nucleus (turquoise, visualized with Hoechst 33342). (H) Western blot analysis of E-cadherin and β-catenin protein expression in control and E-cadherin KO human EBs, as generated by CRISPR/Cas9 editing of mobilized CD34⁺ HSPCs from 3 donors. (I) Representative western blot revealing E-cadherin and β-catenin protein expression in control and E-cadherin KO EBs treated with CHIR99021, a selective GSK3β inhibitor, or dimethyl sulfoxide (DMSO) used as control. (J) Quantification of β-catenin protein expression defined by western blot (n = 3), as normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as input, in control and E-cadherin KO EBs that are treated with DMSO (used as control) or selective GSK3β inhibitor CHIR99021 (n = 3; Student t test; DMSO KO vs CHIR KO: ∗∗P = .002; DMSO WT vs DMSO KO: ∗P = 0.02). IP, intraperitoneal; WT, wild-type.