Extended Data Fig. 3: Impaired B-cell development upon prolonged degradation of E2A, Ebf1, Pax5, and Ikaros.
Flow-cytometric analysis of bone marrow from 6–10-week-old wild-type (Ctrl), Tcf3Aid/Aid, Ebf1Aid/Aid, Pax5Aid/Aid, Ikzf1Aid/Aid, Ikzf3Aid/Aid, and Ikzf1,3Aid/Aid mice expressing the Rosa26Tir1-F74G allele, which were analyzed after three (a,g) or six (f) days, or 18 hours (b-e) upon daily 5-Ph-IAA injection. a,b, The frequencies of ALPs, BLPs (a) and total B cells (b, upper row) are indicated next to the respective gate. The frequency of the CLPs in total bone marrow cells is also shown in (a). One of two independent experiments is shown. Short-term BrdU incorporation (b, lower row) in the last 45 minutes of the 18-hour degradation of the indicated TFs was analyzed by flow cytometry with Vpreb1 [CD179a] staining. The Vpreb1 expression allowed to distinguish between Vpreb1hiB220+CD19+/intIgM–IgD– cells (pro-B and recently generated large pre-B cells), Vpreb1+/intB220+CD19+/intIgM–IgD– cells (large pre-B cells), and Vpreb1–B220+CD19+/intIgM–IgD– cells (small pre-B cells). c, Quantification of bone marrow (BM) and B cells after 18 hours of TF degradation relative to untreated mice of the same genotype (n = 6,3,4,3,4). The gray bar refers to the ratio of two untreated littermates. d, CD19 expression on B cells before and after 18-hours TF degradation (n = 5/4,6/5,4/4,2/4). e, BrdU incorporation in the B220+CD19+/intIgM–IgD– B-cell population comprising pro-B and pre-B cells, based on the flow cytometric data shown in b (bottom row). n = 4/4,5/5,4/4,3/3. f, Impaired B-cell development upon 6-day degradation of Ikaros, Aiolos, or Ikaros plus Aiolos in Ikzf1Aid/Aid, Ikzf3Aid/Aid, and Ikzf1,3Aid/Aid mice, homozygous for Rosa26Tir1-F74G. Bone marrow cells of treated and untreated control mice were analyzed by flow cytometry to determine the frequencies of total B, pro-B, large, small pre-B, and immature B cells (n = 3,3,3,3). g, Developmental arrest at an aberrant large pre-B cell stage upon 3-day degradation of Ikaros and Aiolos. Only IgμhiVpreb1hiKit– B cells were detected by intracellular (i.c.) staining after prolonged protein degradation, while pro-B (Igμ–Vpreb1hiKit+), large pre-B (IgμhiVpreb1hi-intKit–), and small pre-B (IgμintVpreb1–Kit–) cells were identified in untreated mice. Statistical data (c-f) are shown as mean values with SEM and were analyzed by two-way ANOVA with Dunnett’s (c) multiple comparison test and by one-way ANOVA with Å Ãdák’s (d,e) or Dunnett’s (f) multiple comparison tests: **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot (c-f) corresponds to one mouse.
