RhoA is necessary for BAFF-mediated B cell survival but not proliferation.

<p>(A) Splenic B220⁺ B cells from <i>CD19^Cre/+; RhoA^+/+</i> (control) and <i>CD19^Cre/+; RhoA^flox/flox</i> (<i>RhoA^−/−</i>) mice were cultured for 48 hours on 96-well plates (4×10⁵ cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')₂ antibody or LPS. Cell growth rate was analyzed using the CellTiter 96® AQ_ueous Non-Radioactive Cell Proliferation Assay (MTS) kit. Data are expressed as absorbance OD₄₉₀. n = 5. (B) Splenocytes from control and <i>RhoA^−/−</i> mice were stained with anti-B220, -CD21, and -CD23 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (C) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were cultured for 72 hours on 96-well plates (2×10⁵ cells/well) with or without (−) 2 µg/mL anti-IgM F(ab')₂ antibody or indicated concentrations of BAFF (left). Alternatively, control B cells were incubated with or without (−) BAFF and/or Y27632 (10 µM) (right). The cells were then stained with Annexin V and analyzed by flow cytometry. n = 5. (D) Splenocytes from control and <i>RhoA^−/−</i> mice were stained with antibodies against B220, CD21, CD23 and BAFFR, and then analyzed by flow cytometry. The numbers above bracketed lines indicate the percentage of BAFFR⁺ cells in each B cell subset and the numbers below the bracketed lines indicate mean fluorescence intensity (MFI) of BAFFR in each B cell subset (left). The percentage of BAFFR⁺ cells and MFI of BAFFR were averaged from 5 mice for each genotype (right). (E) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were subjected to Western blot for BAFFR and IgM (left). β-actin serves as loading control (left). The protein expression was quantified and normalized to β-actin and the data are expressed as fold of expression (right). n = 3. (F) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were analyzed for BAFFR mRNA levels by quantitative RT-PCR. The expression of GAPDH was used to normalize samples and the relative fold of expression is shown. n = 4. (G) Splenic B220⁺ B cells from control and <i>RhoA^−/−</i> mice were stimulated with or without BAFF(100 ng/mL) for 30 min and then subjected to Western blot (left). Phospho (p)-Akt (S473) is quantified and normalized to total Akt and the data are expressed as relative fold of p-Akt (right). n = 3. Error bars represent mean ± SD. **p<0.01. *p<0.05. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.</p

B cell-specific deletion of RhoA impairs splenic B cell development.

<p>(A) Generation of <i>RhoA^−/−</i> B cells. Left, the loxP/Cre-mediated gene targeting strategy to generate the <i>RhoA</i> knockout allele (<i>RhoA⁻</i>) in B cells. Right, Western blot showing RhoA expression in B220⁺ B cells purified from bone marrow and spleen of <i>CD19^Cre/+; RhoA^+/+</i> (control) and <i>CD19^Cre/+; RhoA^flox/flox</i> (<i>RhoA^−/−</i>) mice. (B) Bone marrow cells from control and <i>RhoA^−/−</i> mice were stained with antibodies against B220 and IgM and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of bone marrow cells by the percentage of each subset of cells (right). n = 5. (C) Splenocytes from control and <i>RhoA^−/−</i> mice were stained with antibodies against B220, CD21 and CD23 and analyzed by flow cytometry (left). The number of B cell subsets was calculated by multiplying the total number of splenocytes by the percentage of each subset of cells (right). T: transitional B cells, FO B: follicular B cells, and MZ B: marginal zone B cells. n = 5. (D) Spleen sections from control and <i>RhoA^−/−</i> mice, stained with hematoxylin and eosin. Data are representative of 3 mice. Error bars represent mean ± SD. **p<0.01. Statistical analysis was performed using a Student's unpaired t-test with a two-tailed distribution.</p

Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR.

<p>Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR.</p

mTOR promotes glycolysis in Lin⁺ cells depending on both mTORC1 and mTORC2.

<p>Bone marrow cells were harvested from the indicated mice. Lin⁺ cells were fractionated from the bone marrow cells and assayed for extracellular acidification rate (ECAR) (A) and Slc2a, PDK1, Hif1α, and HK2 by Quantitative Real-time PCR (B-E). For (B-E), the mRNA expression levels were normalized to one WT mouse. Results are representative of three independent experiments. Error bars represent mean ± SD of 5–8 mice. **<i>P</i> < .01.</p

Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR, Raptor or Rictor.

<p>Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR, Raptor or Rictor.</p

mTOR promotes OXPHOS in Lin^- cells depending on both mTORC1 and mTORC2.

<p>(A) Raptor and Rictor protein expression. Bone marrow cells were harvested from the indicated mice and detected for Raptor and Rictor expression by Western blot. β-actin was blotted as a loading control. (B-F) Bone marrow cells were harvested from the indicated mice. Lin^- cells were fractionated from the bone marrow cells and assayed for oxygen consumption rate (OCR) (B) and Atp5I, Nrf1, Cox5a, and Ndufa32 by Quantitative Real-time PCR (C-F). For (C-F), the mRNA expression levels were normalized to one WT mouse. Results are representative of three independent experiments. Error bars represent mean ± SD of 5–8 mice. **<i>P</i> < .01.</p

Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR or simultaneous deletion of Raptor and Rictor.

<p>Changes in mitochondrial parameters in Lin^- and Lin⁺ cells upon deletion of mTOR or simultaneous deletion of Raptor and Rictor.</p

mTOR promotes energy production in Lin^- cells depending on mTORC1 but inhibits it in Lin⁺ cells independent of mTORC1 and mTORC2.

<p>Bone marrow cells were harvested from the indicated mice. Lin^- (A) and Lin⁺ (B) cells were fractionated from the bone marrow cells and assayed for ATP contents. Results are representative of three independent experiments. Error bars represent mean ± SD of 5–7 mice. **<i>P</i> < .01.</p

mTOR promotes glycolysis in Lin^- cells independent of its kinase activity.

<p>(A) mTOR kinase dead (KD) D2338A mutant knock-in strategy. mTOR KD mutant knock-in was achieved by CRISPR/Cas9 technology. Single guide RNA targeting site (CRISPR target site) is indicated. Amino acid 2338 of mTOR was changed from Asp (D) to Ala (A) (GAC to GCC). In addition, three silent mutations (highlighted in green) were introduced to abolish the restriction enzyme site of Stu I and create the restriction enzyme site of Avr II in the KD allele to facilitate genotyping of the mice (data not shown). The silent mutations also serve to prevent the CRISPR complex from re-cutting the KD allele. (B) Bone marrow cells were harvested from the indicated mice and detected for mTOR and phospho (p)-S6, 4E-BP and Akt by Western blot. Total 4E-BP, S6 and Akt were blotted as loading control. (C-J) Bone marrow cells were harvested from the indicated mice. Lin^- (C, D, G, H) and Lin⁺ (E, F, I, J) cells were fractionated from the bone marrow cells and assayed for oxygen consumption rate (OCR) (C-F) and extracellular acidification rate (ECAR) (G-J). OCR and ECAR profiles are shown in (C, E) and (G, I), respectively. Basal OCR in the absence of oligomycin, FCCP, and antimycin and rotenone from one measurement is shown in (D) and (F). ECAR in the presence of glucose but absence of oligomycin and 2-DG is shown in (H) and (J). Data are representative of three independent experiments. Error bars represent mean ± SD of 5–7 mice. *<i>P</i> < .05, **<i>P</i> < .01 determined by One-way ANOVA followed by Bonferroni test. mTOR^KD: mTOR^loxp/KD;Mx-Cre⁺. NS: no significance.</p

mTOR inhibits OXPHOS in Lin⁺ cells dependent on mTORC1 but not mTORC2.

<p>Bone marrow cells were harvested from the indicated mice. Lin⁺ cells were fractionated from the bone marrow cells and assayed for oxygen consumption rate (OCR) (A) and Atp5I, Nrf1, Cox5a, and Ndufa32 by Quantitative Real-time PCR (B-E). For (B-E), the mRNA expression levels were normalized to one WT mouse. Results are representative of three independent experiments. Error bars represent mean ± SD of 5–8 mice.. **<i>P</i> < .01.</p