β-catenin is not required for RSPO3-induced hematopoietic phenotypes. (A) Schematic depicting the experimental approach to test the effect of RSPO3 overexpression on inducible inactivation of β-catenin in hematopoietic cells. (B) Genomic PCR-based quantification of β-catenin deletion efficiency in various experimental groups. Genomic DNA was collected from sorted CD45⁺ cells 28 days after completion of PolyI:C induction. (C) Representative FACS plots and corresponding bar graph quantifying erythroid progenitors in recipients of autologous bone marrow (BM), Mx1^Cre–β-catenin^fl/fl–derived BM (Mx1Cre^–), or Mx1^Cre+β-catenin^fl/fl–derived BM (Mx1Cre⁺) with or without induction of RSPO3 overexpression; n = 5 to 10 per group. (D) Representative images of hematoxylin and eosin–stained sternum sections showing no reversal of the RSPO3-induced BM phenotype upon loss of β-catenin; n = 5 to 10 per group. (E) Absolute number of RBCs in recipients of autologous BM, Mx1^Cre–β-catenin^fl/fl–derived BM, or Mx1^Cre+β-catenin^fl/fl–derived BM with or without induction of RSPO3 overexpression; n = 5 to 10 per group. (F) Representative FACS plots and corresponding bar graph quantifying early B-progenitors in BM from control and RSPO3 animals of each treatment group; n = 5 to 10 per group. Data shown are mean ± standard error of the mean. One-way analysis of variance followed by Dunnett’s test for multiple comparisons. Scale bar, 500 μm. AutoBMT, autologous bone marrow transplant; BMT, bone marrow transplant; FITC, fluorescein isothiocyanate; IgM, immunoglobulin M; n.s., not significant (P > .05); Tam, tamoxifen.