Fig. 8 | Nature Communications

Fig. 8

From: PLCγ-dependent mTOR signalling controls IL-7-mediated early B cell development

Fig. 8

IL-7-mediated mTOR activation is Akt/Tsc/Rheb pathway-independent. a AKT inhibition does not affect IL-7-dependent B cell production. Wild-type Lin BM cells were cultured on OP9 cells plus IL-7 for 1 day and then treated with DMSO or MK-2206. Numbers indicate percentages of B220+ and Mac-1+ cells in the gated live cells. b AKT inhibition does not block S6 phosphorylation. Pro-B cells derived from Rag1-deficient BM were pre-treated with DMSO, MK-2206 or rapamycin for 10 min and then stimulated with IL-7 for 90 min. Cell lysates were subjected to Western analysis. c Rheb deficiency does not affect early B cell development. The percentages of pro-B cells in the gated BM B220+ cells of Rheb +/+ and Rheb / mice. d IL-7-induced mTOR activation is Rheb-independent. Pro-B cells derived from Rheb +/+ and Rheb / BM were stimulated with IL-7. Cell lysates were subjected to western analysis. e, f mTOR pathway inhibition impairs IL-7-dependent B cell production. Wild-type Lin BM cells were cultured on OP9 cells plus IL-7 for 1 day and then treated with DMSO or rapamycin. Numbers indicate percentages of B220+ and Mac-1+ cells in the gated live cells (e). Bar graphs show the percentages and numbers of B220+ and Mac-1+ cells (f). gi mTOR pathway inhibition blocks or partially impairs IL-7-mediated B cell progenitor proliferation or survival, respectively. Pro-B cells derived from Rag1-deficient BM were cultured without IL-7 (medium) or with IL-7 plus DMSO or rapamycin. Cell proliferation was determined by [3H] thymidine incorporation (g), cell-cycle analysis was performed by PI staining h, and cell survival was measured by TUNEL staining (i). jl mTOR inhibition impairs IL-7-mediated S6 phosphorylation and metabolism in B cell progenitors. Pro-B cells derived from Rag1-deficient BM were stimulated with IL-7 plus or minus rapamycin. Cell lysates were subjected to western analysis (j). These pro-B cells were cultured with IL-7 plus DMSO or rapamycin. After basal OCR (k) and ECAR (l) measurement, oligomycin A (A), FCCP (B), and antimycin (C) were sequentially added. Error bars show ± SEM. Data shown are obtained from or representative of 2 (a, b, d), 3 (c), 5 (e, f, k, l), 6 (g, j), and 7 (h, i) independent experiments

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