Extended Data Fig. 5: Genetic attenuation of β-catenin protein degradation induces acute cell death in pre-B cells in vitro and in vivo.

(a) Pre-B cells from Gsk3b^fl/+ mice were transduced with 4-OHT-inducible Cre-ER^T2 or ER^T2 along with GFP. Western blot analysis was performed to visualize Gsk3b deletion and β-catenin accumulation in GFP⁺ cells 2 days after 4-OHT mediated deletion of one allele of Gsk3b. Effects of Gsk3b haploinsufficiency and β-catenin accumulation were assessed in competitive growth assays monitoring changes in percentages of GFP⁺ cells for 0-8 days after 4-OHT treatment. Mean values from 2 independent experiments each with n = 2 technical replicates are shown. (b) Apc^fl/+ pre-B cells were transduced with GFP-tagged Cre-ER^T2 or ER^T2 constructs, and Western blot analysis was performed in GFP⁺ cells to confirm Apc levels and accumulation of β-catenin 2 days after 4-OHT treatment. Following loss of one allele of APC, competitive fitness of pre-B cells was assayed by measuring changes in percentages of GFP⁺ cells. Data presents mean values from 2 independent experiments with each n = 3 technical replicates. (c) Ck1a^fl/+ pre-B cells were transduced with Cre-ER^T2 or ER^T2 and GFP. Western blot analysis was performed for Ck1α, β-catenin and β-actin 2 days after 4-OHT mediated CK1α deletion. Growth kinetics of cells with monoallelic deletion of Ck1α (Cre-ER^T2) was compared to cells with wild type Ck1α (ER^T2) by flow cytometry of changes in GFP⁺ cells 0-8 days post 4-OHT treatment. Data shows mean values from 2 independent experiments each with n = 2 technical replicates. (d) Pre-B cells from β-catenin^{(S33-S45)+/fl} mice expressing Cre-ER^T2 or ER^T2 along with GFP were treated with 4-OHT to induce Cre-mediated excision of GSK3B-phosphorylation sites. Accumulation of β-catenin was confirmed by Western blot. Removal of exon-3 results in a shorter form of β-catenin. Competitive fitness of pre-B cells was assessed by measuring the frequencies of GFP⁺ cells by FACS. Data are presented as the mean±s.d of 3 independent experiments each with n = 3 technical replicates. (e-h) β-catenin^{(S33-S45)+/fl} mice were crossed with Mb1^Cre/+ mice for B-cell-specific excision of GSK3B-phosphorylation sites and B-cell development in the bone marrow and peripheral lymphoid organs was studied by FACS. (e) Absolute numbers (f) percentages of B-cell precursor subsets in the bone marrow of the mice are shown for Mb1^Cre/+ β-catenin^{(S33-S45)+/fl} (red, n = 6 mice, biological replicates) and Mb1^Cre/+ β-catenin^(S33-S45)+/+ mice (green, n = 6 mice, biological replicates). Bone marrow B-cell precursors were distinguished as pro-B cells (CD43⁺ B220^low IgM⁻ BP1⁻), pre-BI cells (CD43⁺ B220^low IgM⁻ BP1⁺), pre-BII cells (CD43⁻ B220^low, IgM⁻), immature B cells (CD43⁻ B220^low IgM⁺) and mature B cells (CD43⁻ B220^high IgM⁺). (g) Representative FACS plots and absolute numbers and frequencies of B220⁺ B-cells in spleens of Mb1^Cre/+ β-catenin^{(S33-S45)+/fl} (red, n = 5 mice, biological replicates) and Mb1^Cre/+ β-catenin^(S33-S45)+/+ (green, n = 5 mice, biological replicates) mice are shown. (h) Representative FACS plots, absolute numbers, and fractions [%] of B-cells in the peripheral lymph nodes of Mb1^Cre/+ β-catenin^{(S33-S45)+/fl} (red, n = 4 mice, biological replicates) and Mb1^Cre/+ β-catenin^(S33-S45)+/+ (green, n = 4 mice, biological replicates) mice are shown. Throughout (e-h) two tailed unpaired t-tests were used to calculate P values and data are presented as mean±s.d.

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