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. 2018 Nov 22;9:2715. doi: 10.3389/fimmu.2018.02715

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Copyright © 2018 Benhamou, Labi, Getahun, Benchetrit, Dowery, Rajewsky, Cambier and Melamed.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Ablation of miR17-92 in proB cells imposes a mild pro-to-pre B cell block and elevates expression of RAGs. (A) Representative flow cytometry analysis of BM cells from mice with the indicated genotypes (3 mice from each genotype). Initial forward and side scatter gates were set to exclude dead cells and debris. Numbers adjacent to outlined areas indicate % cells amongst total BM cells in each gate. The proB (B220 + IgM- AA4.1 + CD25- ckit+) and preB (B220 + IgM- AA4.1 + CD25 + ckit-) populations are marked with arrows. Also shown are absolute cell counts. (B) The proB and preB cells were quantified for each individual mouse and are expressed as proB/preB ratio. Plot depicts mean from 3 individual mice ± SE. (C) The proB, preB and mature B (B220 + IgM + AA4.1-) cells were sorted from the gates shown in (A) and analyzed for relative expression of PTEN mRNA by qPCR and normalized to Hprt. Results are presented as mean from 3 individual mice ± SE. (D) Intracellular stain for PTEN and pAKT of BM cells gated on B220+/IgM- pro/pre B cells. Graph represents 2 mice in each group. (E) BM culture wild-type pro/preB cells were treated with or without miR19b antagomirs for 48 h and analyzed for the indicated proteins by western blotting. (F) Sorted proB, preB and mature B cells were analyzed for relative expression of RAG-1 (top) and RAG-2 (bottom) by qPCR normalized to Hprt. Graph depicts mean from 3 individual mice ± SE.