Lipopolysaccharide stimulates dynamic changes in B cell metabolism to promote proliferation
- Division of Cell Signalling and Immunology, Faculty of Life Sciences, University of Dundee, United Kingdom
- Department of Pharmacological and Biomolecular Sciences “Rodolfo Paoletti”, University of Milan, Italy
Figures
Figure 1 with 4 supplements
Stimulation with LPS + IL-4 promotes B cell proliferation and class switch recombination.
(A–D) Cells from the lymph nodes of C57BL6/J mice were stained with Cell Trace Violet (CTV) and stimulated with IL-4 (10 ng/ml) +/- LPS (20 μg/ml) for 72 hr and analysed by flow cytometry. Data for naive B cells (stained on day 0) is also shown. Live CD19+B cells were identified using the gating strategy described in Figure 1—figure supplement 4A. (A) Representative flow cytometry plots comparing IgG1 expression and CTV staining after 72 hr of IL-4 or LPS + IL-4 stimulation. (B) Representative plots for FSC and SSC at 72 hr of IL-4 or LPS + IL-4 stimulation with geometric means for the (C) forward scatter and (D) side scatter. Data shows the results of three biological replicates. Data was analysed by one-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparisons to naive B cells, p<0.0001 is indicated by ****. Full ANOVA results are given in Supplementary file 5. (E–H) Cells from the lymph nodes of C57BL6/J mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 hr, then CD19+B cells were isolated by FACS. Alternatively, naive CD19+B cells were sorted directly from ex vivo lymph node cells. Cells were lysed and analysed by proteomics as described in the methods. Samples from four mice for LPS + IL-4 and three mice for naive were generated. (E) Total protein content (pg/cell) was estimated from the proteomic data. Statistical power was determined using an unpaired two-tailed Student’s t-test, where p<0.01 is indicated by **. (F) Volcano plot depicting changes in estimated protein copy number in naive vs LPS + IL-4 stimulated B cells. (G) Volcano plot showing the estimated cellular protein concentration (μM) in naive vs LPS + IL-4 stimulated B cells. Horizontal dashed lines indicate q<0.05. Vertical dashed lines indicate log2 fold change of one standard deviation away from the median. (H) Enrichment analysis of the upregulated proteins in (G) against GO-term and KEGG databases.
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Figure 1—source data 1
Raw FACS files for Figure 1A-D (72 hr LPS + IL-4 stimulation lymph nodes).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig1-data1-v1.zip
Figure 1—figure supplement 1
Unstimulated B cells do not proliferate or undergo class switching.
(A–B) Lymph node cells from C57BL6/J mice were stained with Cell Trace Violet (CTV), cultured for 24 or 72 hr and analysed by flow cytometry. Live CD19+B cells were identified using the gating strategy described in Figure 1—figure supplement 4A. (A) Representative flow cytometry plots comparing IgG1 expression and CTV staining after 24 or 72 hr. (B) Representative plots for FSC and SSC at 24 or 72 hr.
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Figure 1—figure supplement 1—source data 1
Raw FACS files for Figure 1—figure supplement 1A–B (24 vs 72 hr unstimulated lymph nodes).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig1-figsupp1-data1-v1.zip
Figure 1—figure supplement 2
LPS + IL-4 stimulation upregulates glycolysis in B cells.
(A) Volcano plot showing the cellular protein concentration (μM) of proteins involved in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway (PPP) between naive vs LPS + IL-4 stimulated B cells derived from the proteomic dataset described in Figure 1. (B) Volcano plot showing the cellular protein concentration (μM) of proteins involved in the electron transport chain (ECT) between naive vs LPS + IL-4 stimulated B cells. (C) Volcano plot depicting changes in protein copy number of proteins involved in glycolysis, the TCA cycle and PPP between naive vs LPS + IL-4 stimulated B cells. (D) Volcano plot depicting changes in protein copy number of proteins involved in the ECT between naive vs LPS + IL-4 stimulated B cells. For (A–D), horizontal dashed lines indicate q<0.05. Vertical dashed lines indicate log2 fold change of one standard deviation away from the median. (E, F) Graphs depicting changes in cellular concentration (nM) of (E) SLC2A1 and (F) HK2. Adjusted p values from the FDR calculations applied to the proteomic dataset described in Figure 1, where p(adj)<0.05 is indicated by * and p(adj)<0.0001 by ****. (G, H) Splenocytes from C57BL6/J mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 hr before staining with MitoTracker Red to measure mitochondrial volume. Gating strategy described in Figure 1—figure supplement 4B. Representative histograms are shown in (G), and quantification of MFI of MitoTracker staining between naive and LPS + IL-4 stimulated B cells in (H). The graph shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. Statistical power was determined using an unpaired Student’s t-test (two-tailed), where p<0.001 is indicated by ***.
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Figure 1—figure supplement 2—source data 1
Raw FACS files for Figure 1—figure supplement 2G–H (MitoTracker staining).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig1-figsupp2-data1-v1.zip
Figure 1—figure supplement 3
LPS + IL-4 stimulation does not increase STAT1 phosphorylation.
(A) Volcano plot showing the cellular protein concentration (μM) of proteins regulated by IFNα between naive vs LPS + IL-4 stimulated B cells derived from the proteomic dataset described in Figure 1. A list of B-cell-specific genes upregulated by IFNα stimulation from Mostafavi et al., 2016 (Table S1. Transcripts induced by systemic IFNa in different mouse immunocyte lineages). Horizontal dashed lines indicate q<0.05. Vertical dashed lines indicate log2 fold change of one standard deviation away from the median. (B, C) Splenocytes from C57BL6/J mice were stimulated with LPS (20 μg/ml) and/or IFN-β (10 ng/ml) for 15 min before fixing and staining for p-STAT1. Representative histograms are shown in (B), and quantification of the MFI of p-STAT1 staining between naive, IFN-β, LPS, and LPS +IFN-β stimulated B cells in (C). The graph shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis, where p<0.0001 is indicated by ****.
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Figure 1—figure supplement 3—source data 1
Raw FACS files for Figure 1—figure supplement 3B–C (p-STAT1 staining).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig1-figsupp3-data1-v1.zip
Figure 1—figure supplement 4
Representative gating strategies.
(A) Gating strategy for lymph nodes and splenocytes cell size, proliferation, CTV, IgG1 (Figure 1A–D). (B) Gating strategy for MitoTracker (Figure 1—figure supplement 2G–H). (C) Gating strategy for cell cycle analysis (Figure 2A, B, G, H). (D) Gating strategy for OPP uptake (Figure 3H-I, Figure 3—figure supplement 1A-B, Figure 9—figure supplement 2A).
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Figure 1—figure supplement 4—source data 1
Raw FACS files for Figure 1—figure supplement 4A (lymph node gating strategy), 4B (MitoTracker gating strategy), 4 C (cell cycle analysis gating strategy), 4D (OPP gating strategy).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig1-figsupp4-data1-v1.zip
Figure 2
Proteins involved in cell cycle progression are upregulated in LPS + IL-4 activated B cells.
(A–B) B cells were purified from the spleens of C57BL6/J mice and either fixed on isolation (naive) or stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 hr before fixation. Cells were then stained with DAPI and CD19. The cell cycle stages were analysed using the gating strategy shown in Figure 1—figure supplement 4C. (A) Representative histograms showing the proportion of B cells in different phases of the cell cycle. (B) Quantification shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis, where p<0.01 is indicated by **, p<0.001 by *** and p<0.0001 by **** for comparisons between the naive and LPS + IL-4 conditions. (C–F) Graphs depicting changes in the estimated cellular concentration (nM) of proteins implicated in entry into the cell cycle, determined from the proteomic dataset described in Figure 1. (C) Cyclin D, (D) CDK4, (E) CDK6, (F) p27Kip1. (G–H) B cells were purified from the spleens of C57BL6/J mice and stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 hr before fixing and staining for phospho-retinoblastoma (p–Rb). Gating strategy for p-Rb staining is shown in Figure 1—figure supplement 4C. (G) Representative histogram comparing p-Rb staining in naive and LPS + IL-4 stimulated B cells. (H) Quantification shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. (I) Heat map showing the expression of proteins encoded by E2F target genes, derived from the proteomic data. (J–M) Graphs depicting changes in the estimated cellular concentration (nM) of proteins implicated in cell cycle progression, determined from the proteomic dataset described in Figure 1. (J) Cyclin A, (K) Cyclin B, (L) CDK2, (M) CDK1. P values were determined using an unpaired two-tailed Student’s t-test for (H) or represent adjusted p values from the FDR calculations applied to the proteomic dataset (D, E, F, L), where p(adj)<0.01 is indicated by **, p(adj)<0.001 by ***, and p(adj)<0.0001 by ****.
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Figure 2—source data 1
Raw FACS files for Figure 2A–B (DAPI staining) and Figure 2G–H (p-Rb staining).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig2-data1-v1.zip
Figure 3 with 1 supplement
LPS + IL-4 stimulation promotes protein synthesis.
(A, B) Graphs show changes in cellular concentration (nM) of the sum of proteins that make up the large (A) and small (B) ribosomal subunits, with heat maps showing the expression of the individual proteins making up the (C) large and (D) small subunits. Adjusted p values from the FDR calculations applied to the proteomic dataset, where p(adj)<0.001 is indicated by ***. (E–G) The proteomic dataset was mined for proteins involved in the biogenesis of the large and small ribosomal subunits based on the GO terms: GO:0000027, GO:0042273, GO:0000028, GO:0042274. (E) shows the sum of the proteins involved in the biogenesis of the large subunit and (F) shows the small subunit, with individual proteins represented on the volcano plot (G). Horizontal dashed lines on (G) indicate q<0.05 while vertical dashed lines indicate log2 fold change more than one standard deviation away from the median. Adjusted p values from the FDR calculations applied to the proteomic dataset, where p(adj)<0.0001 is indicated by ****. (H–I) Splenocytes from C57BL6/J mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 hr before fixing and staining for the uptake of puromycin analogue O-propargyl-puromycin (OPP) to measure protein synthesis. Gating strategy for OPP staining is shown in Figure 1—figure supplement 4D. (H) Representative histogram comparing OPP uptake between naive and LPS + IL-4 stimulated B cells. (I) Quantification shows three technical replicates from cells isolated from one mouse. Statistical power was determined using an unpaired two-tailed Student’s t-test, where p<0.0001 is indicated by ****.
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Figure 3—source data 1
Raw FACS files for Figure 3H–I (OPP uptake [LPS +IL-4]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig3-data1-v1.zip
Figure 3—figure supplement 1
Protein synthesis is increased in B cells regardless of stimuli.
(A–B) Splenocytes from C57BL6/J mice were plated in normal media before stimulation with LPS (20 μg/ml), Resiquimod (1 μg/ml), ODN 1826 (1 μg/ml), anti-IgM (10 μg/ml), or CD40L (500 ng/ml) for 24 hr before fixing and staining for the uptake of puromycin analogue O-propargyl-puromycin (OPP) to measure protein synthesis. Gating strategy for OPP staining is shown in Figure 1—figure supplement 4D. (A) Representative histogram comparing OPP uptake between different stimuli. (B) Quantification shows the results of three biological replicates. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to naive B cells, p<0.0001 is indicated by ****.
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Figure 3—figure supplement 1—source data 1
Raw FACS files for Figure 3—figure supplement 1A–B (OPP uptake [all agonists]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig3-figsupp1-data1-v1.zip
Figure 4 with 3 supplements
Amino acid transporter SLC7A5 is required for key B cell functions.
(A) Heat map showing the expression of genes encoding for proteins involved in plasma membrane amino acid transport determined from the proteomic dataset described in Figure 1. (B, C) Graphs depicting changes in cellular concentration (nM) of (B) SLC7A5 and (C) SLC3A2 derived from the proteomic data. Adjusted p values from the FDR calculations applied to the proteomic dataset, where p(adj)<0.0001 is indicated by ****. (D–E) Lymph node cells from C57BL6/J mice were stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 hr before staining for CD98. (D) Representative FACS plot comparing CD98 expression. (E) Quantification shows three technical replicates from cells isolated from one mouse and is representative of two independent experiments. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparison to naive B cells, where p<0.001 is indicated by *** and p<0.0001 by ****. (F–I) B cells were purified from the spleens of wild type (WT) and Slc7a5fl/fl/Tg(Vav1-iCre)+/- mice and stained with CTV before stimulation with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 72 hr. (F) Percentage of live B cells (7AAD-ve) in WT and Slc7a5fl/fl/Tg(Vav1-iCre)+/- mice. (G) Histogram representing CTV staining of WT and Slc7a5fl/fl/Tg(Vav1-iCre)+/- B cells. (H) Live B cell number of WT and Slc7a5fl/fl/Tg(Vav1-iCre)+/- mice. (I) Percentage of B cells that are IgG1+ve in WT and Slc7a5fl/fl/Tg(Vav1-iCre)+/- mice. Data shows the results of four biological replicates per genotype. Statistical power was determined using an unpaired two-tailed Student’s t-test, where p<0.0001 is indicated by ****. (J–K) B cells were purified from the spleens of WT and Slc7a5fl/fl/Tg(Vav1-iCre)+/- mice and stimulated with LPS (20 μg/ml) +/- IL-4 (10 ng/ml) for 24 hr before fixing and staining for the uptake of kynurenine to measure amino acid uptake. Gating strategy for kynurenine uptake in Figure 4—figure supplement 3A. (J) Quantification of kynurenine MFI between B cells from WT and Slc7a5fl/fl/Tg(Vav1-iCre)+/- mice with (+) or without (-) LPS +IL-4, kynurenine or aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). (K) Quantification of kynurenine MFI between B cells from WT and Slc7a5fl/fl/Tg(Vav1-iCre)+/- mice with (+) or without (-) LPS, kynurenine or BCH. Data shows the results of four biological replicates per genotype. Statistical power was determined for using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis, where p<0.0001 is indicated by **** for comparisons between genotypes.
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Figure 4—source data 1
Raw FACS files for Figure 4D–E (CD98 staining), 4F-I (WT vs SLC7A5 KO proliferation).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig4-data1-v1.zip
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Figure 4—source data 2
Raw FACS files for Figure 4J (Kynurenine uptake – LPS +IL-4 [WT vs SLC7A5 KO]), 4 K (Kynurenine uptake – LPS [WT vs SLC7A5 KO]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig4-data2-v1.zip
Figure 4—figure supplement 1
LPS + IL-4 stimulation upregulates serine biosynthesis.
(A) Schematic depicting the serine biosynthetic pathway. (B–F) Graphs depicting changes in cellular concentration (nM) of (B) PHGDH, (C) PSAT1, (D) PSPH, (E) SHMT1, and (F) SHMT2. Adjusted p values from the FDR calculations applied to the proteomic dataset described in Figure 1, where p(adj)<0.05 is indicated by *, p(adj)<0.01 by **, p(adj)<0.001 by ***, and p(adj)<0.0001 by ****.
Figure 4—figure supplement 2
Short-term LPS stimulation increases the transcription of genes involved in amino acid uptake and cholesterol metabolism in B cells.
RNA-Seq data comparing reads per kilobase million (RPKM) between naive and LPS stimulated B cells at different time points from wild-type C57BL/6 mice derived from dataset EV1 (Tesi et al., 2019). Reads per kilobase million (RPKM) of (A) SLC7A5, (B) SLC3A2, (C) HMGCR, (D) SQLE, (E) LDLR, and (F) SREBP2. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to naive B cells, p<0.01 is indicated by **, p<0.001 by ***, and p<0.0001 by ****. ns indicated by p>0.05.
Figure 4—figure supplement 3
Representative gating strategies.
(A) Gating strategy for kynurenine uptake (Figure 4J-K, Figure 9—figure supplement 2B). (B) Gating strategy for filipin staining (Figures 5F-I and 7A-B, Figures 8I-J and 9F, Figure 10A-B, Figure 5—figure supplement 2J, Figure 7—figure supplement 1). (C) Gating strategy for purified B cells for cell size, proliferation, and CTV (Figures 6A-P and 7C-F, Figures 8A-H and 9A-E, Figure 10C-E, Figure 5—figure supplement 2A-I).
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Figure 4—figure supplement 3—source data 1
Raw FACS files for Figure 4—figure supplement 3A (Kynurenine uptake gating strategy), 3B (filipin staining gating strategy), 3 C (purified B cell gating strategy).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig4-figsupp3-data1-v1.zip
Figure 5 with 2 supplements
LPS + IL-4 stimulation upregulates cholesterol metabolism in B cells.
(A–E) Analysis of the genes involved in cholesterol metabolism in the proteomic dataset. (A) Heat map of all the enzymes involved in cholesterol biosynthesis. Cellular concentration (nM) of (B) HMGCR, (C) SQLE, (D) LDLR, and (E) SREBP2. Adjusted p values from the FDR calculations applied to the proteomic dataset, where p(adj)<0.01 is indicated by **. (F) Splenocytes from C57BL6/J mice were plated in cholesterol-free (CF) media and stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml). The cells were fixed at the stated time points and stained with filipin. The gating strategy for filipin staining in Figure 4—figure supplement 3B. (F) Filipin time course. Data shows three technical replicates from cells isolated from one mouse. Statistical power was determined by one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to naive B cells, p<0.05 is indicated by * and p<0.0001 by ****. ns indicated by p>0.05. (G–H) Splenocytes from C57BL6/J mice were plated in cholesterol-free media and pre-treated with DMSO as a vehicle control or varying concentrations of Fluvastatin or NB-598 for 45 min before stimulation with LPS (20 μg/ml) and IL-4 (10 ng/ml). The cells were fixed after 24 hr and stained with filipin before acquisition. (G) Filipin staining of Fluvastatin titration. (H) Filipin staining of NB-598 titration. Data shows three technical replicates from cells isolated from one mouse and is representative of two independent experiments, each with one biological replicate. Statistical power was determined using a one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparison to LPS + IL-4 stimulated B cells, p<0.001 is indicated by *** and p<0.0001 by ****. (I) Splenocytes from C57BL6/J mice were plated in normal or cholesterol-free (CF) media and pre-treated with DMSO as a vehicle control, Fluvastatin (10 μM) or NB-598 (10 μM) before stimulation with LPS +IL-4. The cells were fixed after 24 hr and stained with filipin before acquisition. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis. For comparisons to the LPS + IL-4 condition, p<0.0001 is indicated by ****.
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Figure 5—source data 1
Raw FACS files for Figure 5F (filipin staining timecourse), 5 G (filipin staining – Fluvastatin titration).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig5-data1-v1.zip
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Figure 5—source data 2
Raw FACS files for Figure 5H (filipin staining – NB-598 titration), 5I (filipin staining – Fluvastatin/NB-598 treatment (normal/CF)).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig5-data2-v1.zip
Figure 5—figure supplement 1
Terpenoid and cholesterol biosynthetic pathway.
Schematic depicting the terpenoid and cholesterol biosynthetic pathways.
Figure 5—figure supplement 2
Blocking rate-limiting enzymes in the cholesterol biosynthesis pathway reduces B cell growth, survival, and proliferation.
(A–I) B cells were purified from the spleens of C57BL6/J mice and cultured in normal media. The cells were stained with CTV and pre-treated with DMSO as a vehicle control or varying concentrations of Fluvastatin, NB-598 or Rosuvastatin for 45 min before stimulation with LPS (20 μg/ml) and IL-4 (10 ng/ml) for 24 hr. For all panels, cells in the absence of LPS + IL-4 were naive B cells analysed on the day of isolation. (A–C) relates to the data in Figure 5G and shows the (A) percentage of live (7AAD-ve) B cells, (B) forward scatter of B cells and (C) representative histograms for CTV staining for Fluvastatin treatment. (D–F) relates to the data in Figure 5H and shows the (D) percentage of live (7AAD-ve) B cells, (E) forward scatter of B cells, and (F) representative histograms for CTV staining for NB-598 treatment. (G–I) Graphs show the (G) percentage of live (7AAD-ve) B cells, (H) forward scatter of B cells, and (I) representative histograms for CTV staining for Rosuvastatin treatment. (J) Splenocytes from C57BL6/J mice were plated in cholesterol-free (CF) media and pre-treated with DMSO as a vehicle control or varying concentrations of Rosuvastatin for 45 min before stimulation with LPS (20 μg/ml) and IL-4 (10 ng/ml). The cells were fixed after 24 hr and stained with filipin. (J) Filipin staining of Rosuvastatin titration. Graphs show three technical replicates from cells isolated from one mouse and are representative of three independent experiments. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis, where p<0.05 is indicated by *, p<0.01 by **, p<0.001 by ***, and p<0.0001 by **** for comparison to the LPS + IL-4 condition. ns indicated by p>0.05.
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Figure 5—figure supplement 2—source data 1
Raw FACS files for Figure 5—figure supplement 2A–C (Fluvastatin titration – 24 hr LPS + IL-4 stimulation), 2D-F (NB-598 titration – 24 hr LPS + IL-4 stimulation).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig5-figsupp2-data1-v1.zip
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Figure 5—figure supplement 2—source data 2
Raw FACS files for Figure 5—figure supplement 2G–I (Rosuvastatin titration – 24 hr LPS + IL-4 stimulation), 2 J (filipin staining – Rosuvastatin titration).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig5-figsupp2-data2-v1.zip
Figure 6 with 1 supplement
Blocking rate-limiting enzymes in the cholesterol biosynthesis pathway reduces B cell growth, survival, and proliferation.
(A–D) B cells were purified from the spleens of C57BL6/J mice and cultured in normal or cholesterol-free (CF) media. The cells were stained with CTV, then pre-treated with DMSO as a vehicle control, Fluvastatin (10 µM), or NB-598 (10 µM), where indicated, for 45 min prior to stimulation with LPS (20 μg/ml) and IL-4 (10 μg/ml) for 48 hr. Gating strategy for CTV staining is shown in Figure 4—figure supplement 3C. (A) shows representative CTV staining, (B) live B cell number, (C) percentage of live B cells (7AAD-ve), and (D) forward scatter. Data shows the results of three biological replicates. (E–H) Same as (A–D) but with pretreatment using FGTI-2734 (10 µM). (E) shows representative CTV staining, (F) live B cell number, (G) percentage of live B cells (7AAD-ve), and (H) forward scatter of B cells. Data shows the results of three biological replicates. (I–P) Same as (A–D) but with pretreatment using FTI-277 or GGTI-298 (10 µM-30 µM). (I) shows representative CTV staining in normal media, (J) live B cell number, (K) percentage of live B cells (7AAD-ve), and (L) forward scatter of B cells. (M) shows representative CTV staining in CF media, (N) live B cell number, (O) percentage of live B cells (7AAD-ve), and (P) forward scatter of B cells. Data shows the results of three biological replicates. (B–D) (F–H) Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis. For comparisons to the LPS + IL-4 condition, p<0.05 is indicated by *, p<0.01 by **, p<0.001 by *** and p<0.0001 by ****. ns indicated by p>0.05. (J–L) (N–P) Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to the LPS + IL-4 condition, p<0.01 is indicated by **, p<0.001 by *** and p<0.0001 by ****. ns indicated by p>0.05. For all panels, cells in the absence of LPS + IL-4 were naive B cells analysed on the day of isolation.
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Figure 6—source data 1
Raw FACS files for Figure 6A–D (proliferation – Fluvastatin/NB-598 treatment [normal/CF]), 6E-H (proliferation – FGTI-2734 treatment [normal/CF]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig6-data1-v1.zip
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Figure 6—source data 2
Raw FACS files for Figure 6I–L (GGTI-298 treatment [normal/CF]), 6 M-P (proliferation – FTI-277 [normal/CF]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig6-data2-v1.zip
Figure 6—figure supplement 1
Blocking cholesterol biosynthesis or uptake upregulates proteins involved in cholesterol metabolism in B cells.
B cells were purified from the spleens of C57BL6/J mice and cultured in normal or cholesterol-free (CF) media. The cells were then pre-treated with DMSO as a vehicle control or Fluvastatin (10 µM), where indicated, for 45 min prior to stimulation with LPS (20 μg/ml) and IL-4 (10 μg/ml) for 24 hr. Alternatively, naive B cells were lysed directly. Cells were lysed and analysed by proteomics as described in the methods. Samples from four mice for each condition were generated. (A) Volcano plot showing the estimated cellular protein concentration (μM) of LPS + IL-4 stimulated B cells cultured in normal or CF media. (B) Volcano plot showing the estimated cellular protein concentration (μM) of LPS + IL-4 stimulated B cells cultured in normal media +/- Fluvastatin. (C) Volcano plot showing the estimated cellular protein concentration (μM) of LPS + IL-4 stimulated B cells cultured in normal media or CF media + Fluvastatin. Horizontal dashed lines indicate q<0.01. Vertical dashed lines indicate log2 fold change of one standard deviation away from the median. Heat map showing the expression of proteins significantly (D) upregulated or (E) downregulated derived from the proteomic data. (F) Heatmap of all the enzymes involved in cholesterol biosynthesis. (G–I) Graphs depicting changes in cellular concentration (nM) of (G) HMGCR, (H) SQLE, and (I) LDLR. Statistical power was determined using a one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparison to the LPS + IL-4 condition, where p<0.01 is indicated by **, p<0.001 by ***, p<0.0001 by ****.
Figure 7 with 1 supplement
Mevalonate supplementation rescues the effect of Fluvastatin treatment in B cells.
(A–B) Splenocytes from C57BL6/J mice were plated in normal or cholesterol-free (CF) media and pre-treated with HEPES as a vehicle control or mevalonate (2 mM) for 1 hr prior to treatment with DMSO or Fluvastatin (10 µM), where indicated, for 45 min before stimulation with LPS (20 μg/ml) and IL-4 (10 ng/ml). The cells were fixed after 24 hr or 48 hr of LPS + IL-4 stimulation and stained with filipin. (A) Filipin staining comparing B cells +/- Fluvastatin or mevalonate after 24 or 48 hr of LPS + IL-4 stimulation in normal media. (B) Filipin staining comparing B cells +/-Fluvastatin or mevalonate after 24 or 48 hr of LPS + IL-4 stimulation in CF media. Data shows the results of three biological replicates. (C–F) B cells were purified from the spleens of C57BL6/J mice and cultured in normal or cholesterol-free (CF) media. The cells were stained with CTV, then pre-treated with HEPES as a vehicle control or mevalonate (2 mM) for 1 hr. The cells were treated with DMSO or Fluvastatin (10 µM), where indicated, for 45 min prior to stimulation with LPS (20 μg/ml) and IL-4 (10 μg/ml) for 48 hr. (C) Shows percentage of live B cells (7AAD-ve), (D) forward scatter of B cells, (E) representative CTV staining, and (F) live B cell number. Data shows the results of three biological replicates. Where shown, statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparison to Fluvastatin-treated B cells, p<0.05 is indicated by *, p<0.01 by **, p<0.001 by ***, p<0.0001 by ****. ns by >0.05. For all panels, cells in the absence of LPS + IL-4 were naive B cells analysed on the day of isolation.
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Figure 7—source data 1
Raw FACS files for Figure 7A (filipin staining – mevalonate supplementation [normal]), 7B (filipin staining - mevalonate supplementation [CF]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig7-data1-v1.zip
-
Figure 7—source data 2
Raw FACS files for Figure 7C–F (proliferation – mevalonate supplementation [normal/CF]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig7-data2-v1.zip
Figure 7—figure supplement 1
High levels of exogenous mevalonate are toxic to B cells.
Splenocytes from C57BL6/J mice were plated in normal media and pre-treated with HEPES as a vehicle control or mevalonate at the stated concentrations for 1 hr prior to LPS (20 μg/ml) and IL-4 (10 ng/ml) stimulation. The cells were cultured for 24 hr, then fixed and stained with filipin prior to acquisition. Gating strategy described in Figure 4—figure supplement 3B. (A) Filipin staining. (B) Percentage of live B cells (L/D-ve). The graph shows three technical replicates from cells isolated from one mouse and is representative of two independent experiments. Statistical power for was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis, where p<0.0001 is indicated by ****.
-
Figure 7—figure supplement 1—source data 1
Raw FACS files for Figure 7—figure supplement 1A–B (Mevalonate toxicity).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig7-figsupp1-data1-v1.zip
Figure 8
GGPP supplementation rescues the effect of Fluvastatin treatment in B cells.
(A–H) B cells were purified from the spleens of C57BL6/J mice and cultured in normal or cholesterol-free (CF) media. The cells were stained with CTV, pre-treated with methanol:ammonium hydroxide solution (CH3OH:NH4OH) as a vehicle control, mevalonate (2 mM), or geranylgeranyl pyrophosphate (GGPP; 10 μM) for 1 hr before treatment with Fluvastatin (10 μM), where indicated. The cells were stimulated with LPS (20 μg/ml) and IL-4 (10 μg/ml) and cultured for 48 hr. (A) shows representative CTV staining for B cells cultured in normal media, (B) live B cell number, (C) percentage of live B cells, and (D) forward scatter of B cells. (E) shows representative CTV staining for B cells cultured in CF media, (F) live B cell number, (G) percentage of live B cells, and (H) forward scatter of B cells. Data shows the results of three biological replicates. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparison to Fluvastatin treated B cells, p<0.001 is indicated by *** and p<0.0001 by ****. ns indicated p>0.05. For (B) and (F), this data was log-transformed and then statistically analysed due to unequal variance. (I–J) Splenocytes were plated in normal or cholesterol-free (CF) media and pre-treated with methanol:ammonium hydroxide solution (CH3OH:NH4OH) as a vehicle control or geranylgeranyl pyrophosphate (GGPP; 10 μM), where indicated, for 1 hr before treatment with DMSO or Fluvastatin (10 μM). The cells were then stimulated with LPS (20 μg/ml) and IL-4 (10 ng/ml). The cells were fixed after 24 hr and stained with filipin before acquisition. (I) Filipin staining comparing B cells +/- Fluvastatin or GGPP after 24 or 48 hr of LPS + IL-4 stimulation in normal media. (J) Filipin staining comparing B cells +/-Fluvastatin or GGPP after 24 or 48 hr of LPS + IL-4 stimulation in CF media. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparison to Fluvastatin-treated B cells, p<0.01 is indicated by **, p<0.001 by ***, and p<0.0001 by ****. ns indicated p>0.05. For all panels, cells in the absence of LPS + IL-4 were naive B cells analysed on the day of isolation.
-
Figure 8—source data 1
Raw FACS files for Figure 8A–D (proliferation – GGPP supplementation [normal]), 8E-H (proliferation – GGPP supplementation [CF]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig8-data1-v1.zip
-
Figure 8—source data 2
Raw FACS files for Figure 8I (filipin staining – GGPP supplementation [normal]), 8 J (filipin staining – GGPP supplementation [CF]).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig8-data2-v1.zip
Figure 9 with 2 supplements
MAPK and mTOR signalling regulate B cell proliferation and cholesterol levels.
(A–E) B cells were purified from the spleens of C57BL6/J mice and stained with CTV, then pre-treated with DMSO as a vehicle control, PD18352 (2 μM), VX745 (1 μM), or Rapamycin (20 nM), where indicated, for 45 min prior to stimulation with LPS (20 μg/ml) and IL-4 (10 μg/ml) for 48 hr. (A) shows representative CTV staining, (B) percentage of B cells per generation quantified from (A), (C) live B cell number (D) percentage of live B cells (7AAD-ve), and (E) forward scatter. Data shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. Statistical power for (B) was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis. For comparison to LPS + IL-4 stimulated B cells, p<0.0001 by **** and ns by >0.05. (F) Splenocytes from C57BL6/J mice were pre-treated with DMSO as a vehicle control, PD18352 (2 μM), VX745 (1 μM), or Rapamycin (20 nM), where indicated, for 45 min prior to stimulation with LPS (20 μg/ml) and IL-4 (10 μg/ml). The cells were fixed after 24 hr and stained with filipin before acquisition. (F) Filipin staining after inhibitor treatment. Data shows three technical replicates from cells isolated from one mouse and is representative of three independent experiments. Statistical power for (C-F) was determined by one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis, where p<0.05 is indicated by *, p<0.01 by **, p<0.001 by ***, p<0.0001 by **** and ns by >0.05. For all panels, cells in the absence of LPS + IL-4 were naive B cells analysed on the day of isolation.
-
Figure 9—source data 1
Raw FACS files for Figure 9A–E (proliferation – PD184352, VX745, Rapamycin), 9 F (filipin staining - PD184352, VX745, Rapamycin).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig9-data1-v1.zip
Figure 9—figure supplement 1
MyD88 is required for signalling through TLR4.
B cells were purified from the spleens of WT and MyD88-/- (MyD88 KO) mice, then stimulated with LPS (20 μg/ml). The cells were lysed after 30 min. Western blotting was used to probe samples with the appropriate antibody (Table 4).
-
Figure 9—figure supplement 1—source data 1
PDF files containing labelled and uncropped images for western blots displayed in Figure 9—figure supplement 1.
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig9-figsupp1-data1-v1.zip
-
Figure 9—figure supplement 1—source data 2
Original files for western blot images displayed in Figure 9—figure supplement 1.
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig9-figsupp1-data2-v1.zip
Figure 9—figure supplement 2
Inhibition of mTOR impacts B cell function.
(A) Splenocytes from C57BL6/J mice were pre-treated with DMSO as a vehicle control or rapamycin (20 nM), where indicated, for 45 min. The cells were stimulated with LPS (20 μg/ml) and IL-4 (10 μg/ml) for 24 hr before fixing and staining for the uptake of puromycin analogue O-propargyl-puromycin (OPP) to measure protein synthesis. Gating strategy for OPP staining is shown in Figure 1—figure supplement 4D. (A) OPP uptake after rapamycin treatment. Data shows the results of three biological replicates. (B) B cells were purified from the spleens of C57BL6/J mice, then pre-treated with DMSO as a vehicle control or rapamycin (20 nM), where indicated, for 45 min. The cells were stimulated with LPS (20 μg/ml) and IL-4 (10 μg/ml) for 24 hr before fixing and staining for the uptake of kynurenine to measure amino acid uptake. Gating strategy for kynurenine uptake in Figure 4—figure supplement 3A. (B) Kynurenine uptake after rapamycin treatment. Data shows the results of three biological replicates. Statistical power was determined by one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis, where p<0.05 is indicated by *, p<0.001 by *** and p<0.0001 by ****.
-
Figure 9—figure supplement 2—source data 1
Raw FACS files for Figure 9—figure supplement 2A (OPP uptake – rapamycin), 2B (kynurenine uptake – rapamycin).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig9-figsupp2-data1-v1.zip
Figure 10 with 1 supplement
Cholesterol is required for the growth and proliferation of B cells by multiple stimuli.
(A) Splenocytes from C57BL6/J mice were plated in normal or cholesterol-free (CF) media, before stimulation with LPS (20 μg/ml), IL-4 (10 μg/ml), or a combination of LPS and IL-4. The cells were fixed after 24 hr and stained with filipin. (A) Filipin staining comparing cholesterol content between different stimuli. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis. For comparisons to naive B cells, p<0.0001 is indicated by ****. (B) Splenocytes from C57BL6/J mice were plated in normal media before stimulation with LPS (20 μg/ml), Resiquimod (1 μg/ml), ODN 1826 (1 μg/ml), anti-IgM (10 μg/ml), or CD40L (500 ng/ml). The cells were fixed after 24 hr and stained with filipin. (B) Filipin staining comparing cholesterol content between different stimuli. Data shows the results of three biological replicates. Statistical power was determined using one-way ANOVA followed by multiple comparison testing via Dunnett’s analysis. For comparisons to naive B cells, p<0.0001 is indicated by ****. (C–E) B cells were purified from the spleens of C57BL6/J mice and cultured in normal media. The cells were stained with CTV, then pre-treated with DMSO as a vehicle control or Fluvastatin (10 µM) for 45 min before stimulation with LPS (20 μg/ml), Resiquimod (1 μg/ml), ODN 1826 (1 μg/ml), anti-IgM (10 μg/ml), or CD40L (500 ng/ml) as indicated for 72 hr. (C) shows the percentage of live B cells (7AAD-ve), (D) representative histogram for CTV staining, and (E) live B cell number. Data shows the results of three biological replicates. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Tukey’s analysis, where p<0.0001 is indicated by ****. For (E), this data was log-transformed and then statistically analysed due to unequal variance. Statistical power was determined using two-way ANOVA followed by multiple comparison testing via Sidak’s analysis, where p<0.0001 is indicated by ****.
-
Figure 10—source data 1
Raw FACS files for Figure 10A (filipin staining – IL-4, LPS, LPS +IL-4 [normal/CF]), 10B (filipin staining – all agonists).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig10-data1-v1.zip
-
Figure 10—source data 2
Raw FACS files for Figure 10C–D (proliferation – all agonists).
- https://cdn.elifesciences.org/articles/109093/elife-109093-fig10-data2-v1.zip
Figure 10—figure supplement 1
IL-4, anti-IgM, and anti-CD40 stimulation upregulate proteins involved in cholesterol metabolism in B cells.
Proteomic data comparing protein concentration (nM) between naive, IL-4, anti-IgM, or anti-CD40 stimulated B cells for 40 hr from wild-type C57BL/6 mice derived from James et al., 2026. (A–D) Graphs depicting changes in cellular concentration (nM) of (A) HMGCR, (B) SQLE, (C) LDLR, and (D) SREBP2.
Tables
Table 1
MACS antibodies.
| Antibody | Clone | Concentration | Catalogue number | Supplier |
|---|---|---|---|---|
| Anti-CD11b | M1/70 | 10 µg/ml | 101204 | BioLegend |
| Anti-CD11c | N418 | 10 µg/ml | 117304 | BioLegend |
| Anti-CD3ε | 145–2 C11 | 10 µg/ml | 100304 | BioLegend |
| Anti-TER119 | TER-119 | 10 µg/ml | 116204 | BioLegend |
Table 2
Inhibitors.
| Name | Target | Concentration | Catalogue number | Supplier |
|---|---|---|---|---|
| 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) | LAT1 | 10 mM | A7902-1G | Sigma |
| Fluvastatin sodium | HMG-CoA reductase | 0.1 µM –100 µM | 3309 | Tocris |
| Rosuvastatin calcium | HMG-CoA reductase | 0.1 µM –100 µM | 6343 | Tocris |
| NB-598 | Squalene monooxygenase | 0.1 µM –100 µM | HY-16343 | MedChem Express |
| FGTI-2734 | Farnesyl and geranylgeranyl transferase-1 | 10 µM | HY-128350 | MedChem Express |
| FTI-277 hydrochloride | Farnesyl transferase | 10 µM-30µM | HY-15872A | MedChem Express |
| GGTI-289 trifluoroacetate | Geranylgeranyl transferase-1 | 10 µM-30µM | HY-15871 | MedChem Express |
| PD18352 | MSK1/2 | 2 µM | 4237 | Tocris |
| VX745 | P38 | 1 µM | 3915 | Tocris |
| Rapamycin | mTOR | 20 nM | R8781 | Merck |
Table 3
Flow cytometry antibodies.
| Antibody | Fluorophore | Clone | Dilution | Catalogue number | Supplier |
|---|---|---|---|---|---|
| Anti-CD19 | APC | 6D5 | 1:300 | 115512 | Biolegend |
| Anti-CD19 | PE | 1D3 | 1:200 | 557399 | BD Biosciences |
| Anti-CD19 | FITC | 1D3 | 1:200 | 152403 | BioLegend |
| Anti-CD93 | APC | AA4.1 | 1:200 | 136510 | BioLegend |
| Anti-CD98 | PE | RL388 | 1:200 | 128208 | BioLegend |
| dsDNA | eFluor 660 | - | 1:1000 | 65-0864-14 | eBioscience |
| Anti-IgG1 | FITC | A85-1 | 1:200 | 553443 | BD Biosciences |
| Anti-pRb | FITC | D20B12 | 1:300 | 4277 S | Cell Signaling Technology |
Table 4
Western blot primary antibodies.
| Antibody target | Animal raised in | Dilution | Code | Source |
|---|---|---|---|---|
| Phospho-STAT1 (Tyr701) | Rabbit | 1:1000 | 9167 | Cell Signalling Technology |
| Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) | Rabbit | 1:1000 | 9101 | Cell Signalling Technology |
| p44/42 MAPK (Erk1/2) | Rabbit | 1:1000 | 9102 | Cell Signalling Technology |
| Phospho-p38 MAPK (Thr180/Tyr182) | Rabbit | 1:1000 | 4511 | Cell Signalling Technology |
| p38 MAPK | Rabbit | 1:1000 | 9212 | Cell Signalling Technology |
| Phospho-Akt (Ser473) | Rabbit | 1:1000 | 4060 | Cell Signalling Technology |
| Phospho-p70 S6 Kinase (Thr389) | Rabbit | 1:1000 | 9234 | Cell Signalling Technology |
| Phospho-S6 Ribosomal Protein (Ser235/236) | Rabbit | 1:1000 | 2211 | Cell Signalling Technology |
| S6 Ribosomal Protein (5G10) | Rabbit | 1:1000 | 2217 | Cell Signalling Technology |
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | C57BL/6J (WT) | Charles River UK | ||
| Genetic reagent (Mus musculus) | Slc7a5fl/fl/Tg(Vav1-iCre)+/- | Poncet et al., 2014 | ||
| Genetic reagent (Mus musculus) | MyD88−/− | Adachi et al., 1998 | ||
| Biological sample, Escherichia coli O26:B6 | Lipopolysaccharide | Sigma Aldrich | Catalogue number: L2654 | Concentration: 20 µg/ml |
| Biological sample, Streptomyces filipinensis | Filipin | Merck | Catalogue number: F9765 | Concentration: 1 µg/µl |
| Antibody | Anti-mouse/human CD11b (Rat, monoclonal, M1/70) | BioLegend | Catalogue number: 101204 RRID:AB_312787 | Concentration: 10 µg/ml |
| Antibody | Anti-mouse CD11c (Armenian hamster, monoclonal, N418) | BioLegend | Catalogue number: 117304 RRID:AB_313773 | Concentration: 10 µg/ml |
| Antibody | Anti-mouse CD3ε (Armenian hamster, monoclonal, 145–2 C11) | BioLegend | Catalogue number: 100304 RRID:AB_312669 | Concentration: 10 µg/ml |
| Antibody | Anti-mouse TER119 (Rat, monoclonal, TER-119) | BioLegend | Catalogue number: 116204 RRID:AB_313705 | Concentration: 10 µg/ml |
| Antibody | Anti-mouse CD19 (Rat, monoclonal, 6D5) | BioLegend | Catalogue number: 115512 RRID:AB_313647 | Fluorophore: APC Cell surface stain dilution: (1:300) |
| Antibody | Anti-mouse CD19 (Rat, monoclonal, 1D3) | BD Biosciences | Catalogue number: 557399 RRID:AB_396682 | Fluorophore: PE Cell surface stain dilution: (1:200) |
| Antibody | Anti-mouse CD19 (Rat, monoclonal, 1D3) | BioLegend | Catalogue number: 152403 RRID:AB_2629812 | Fluorophore: FITC Cell surface stain dilution: (1:200) |
| Antibody | Anti-mouse CD93 (Rat, monoclonal, AA4.1) | BioLegend | Catalogue number: 136510 RRID:AB_2275868 | Fluorophore: APC Cell surface stain dilution: (1:200) |
| Antibody | Anti-mouse CD98 (Rat, monoclonal, RL388) | BioLegend | Catalogue number: 128208 RRID:AB_2190813 | Fluorophore: PE Cell surface stain dilution: (1:200) |
| Antibody | Anti-mouse IgG1 (Rat, monoclonal, A85-1) | BD Biosciences | Catalogue number: 553443 RRID:AB_394862 | Fluorophore: FITC Cell surface stain dilution: (1:200) |
| Antibody | Phospho-Rb (Ser807/811) (Rabbit, monoclonal, D20B12) | Cell Signalling Technology | Catalogue number: 4277 RRID:AB_2797605 | Fluorophore: Alexa Fluor 488 Intracellular stain dilution: (1:300) |
| Antibody | Anti-rabbit IgG (H+L), F(ab')2 Fragment (Goat, unknown clonality) | Cell Signalling Technology | Catalogue number: 4414 RRID:AB_10693544 | Fluorophore: Alexa Fluor 647 Conjugate Dilution: (1:1000) |
| Antibody | Phospho-STAT1 (Tyr701) (Rabbit, monoclonal, 58D6) | Cell Signalling Technology | Catalogue number: 9167 RRID:AB_561284 | Intracellular stain dilution: (1:1000) |
| Antibody | Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Rabbit, polyclonal) | Cell Signalling Technology | Catalogue number: 9101 RRID:AB_331646 | Western blot dilution: (1:1000) |
| Antibody | p44/42 MAPK (Erk1/2) (Rabbit, polyclonal) | Cell Signalling Technology | Catalogue number: 9102 RRID:AB_330744 | Western blot dilution: (1:1000) |
| Antibody | Phospho-p38 MAPK (Thr180/Tyr182) (Rabbit, monoclonal, D3F9) | Cell Signalling Technology | Catalogue number: 4511 RRID:AB_2139682 | Western blot dilution: (1:1000) |
| Antibody | p38 MAPK (Rabbit, polyclonal) | Cell Signalling Technology | Catalogue number: 9212 RRID:AB_330713 | Western blot dilution: (1:1000) |
| Antibody | Phospho-Akt (Ser473) (Rabbit, monoclonal, D9E) | Cell Signalling Technology | Catalogue number: 4060 RRID:AB_2315049 | Western blot dilution: (1:1000) |
| Antibody | Phospho-p70 S6 Kinase (Thr389) (Rabbit, monoclonal, 108D2) | Cell Signalling Technology | Catalogue number: 9234 RRID:AB_2269803 | Western blot dilution: (1:1000) |
| Antibody | Phospho-S6 Ribosomal Protein (Ser235/236) (Rabbit, polyclonal) | Cell Signalling Technology | Catalogue number: 2211 RRID:AB_331679 | Western blot dilution: (1:1000) |
| Antibody | S6 Ribosomal Protein (Rabbit, monoclonal, 5G10) | Cell Signalling Technology | Catalogue number: 2217 RRID:AB_331355 | Western blot dilution: (1:1000) |
| Antibody | Anti-Mouse IgM μ chain (Goat, polyclonal) | Stratech | Catalogue number: 115-006-075 RRID:AB_2338474 | Concentration: 10 µg/ml |
| Peptide, recombinant protein | IL-4 | Peprotech | Catalogue number: 214–14 | Concentration: 10 ng/ml |
| Peptide, recombinant protein | Resiquimod/R848 | Invivogen | Catalogue number: tlrl-r848 | Concentration:1 µg/ml |
| Peptide, recombinant protein | Bléry et al., 2006 | Invivogen | Catalogue number: tlrl-2006 | Concentration:1 µg/ml |
| Peptide, recombinant protein | CD40 ligand | R&D | Catalogue number: 8230 CL-050/CF | Concentration: 500 ng/ml |
| Chemical compound, drug | O-propargyl-puromycin | Jena Bioscience | Catalogue number: NU-931–5 | Concentration: 20 μM |
| Chemical compound, drug | Saponin | Sigma Aldrich | Catalogue number: SAE0073 | Concentration: 0.01% |
| Chemical compound, drug | Copper sulphate | Sigma Aldrich | Catalogue number: 209198 | Concentration: 1 mM |
| Chemical compound, drug | Sodium ascorbate | Sigma Aldrich | Catalogue number: A7631 | Concentration: 10 mM |
| Chemical compound, drug | BTTAA | Sigma Aldrich | Catalogue number: 906328 | Concentration: 1 mM |
| Chemical compound, drug | Aminoguanidine | Cayman Chemicals | Catalogue number: 81530 | Concentration: 10 mM |
| Chemical compound, drug | Alexafluor 647 azide | Invitrogen | Catalogue number: A10277 | Concentration: 5 µM |
| Chemical compound, drug | L-Kynurenine | Sigma Aldrich | Catalogue number: K8625 | Concentration: 200 μM |
| Chemical compound, drug | 2-Amino-2-norbornanecarboxylic acid (BCH) | Sigma Aldrich | Catalogue number: A7902-1G | Concentration: 10 mM |
| Chemical compound, drug | Mevalonic acid 5-phosphate lithium salt hydrate | Sigma Aldrich | Catalogue number: 79849 | Concentration: 1 mM-2mM |
| Chemical compound, drug | Geranylgeranyl pyrophosphate ammonium salt | Sigma Aldrich | Catalogue number: G6025 | Concentration: 10 µM |
| Chemical compound, drug | Fluvastatin sodium | Tocris | Catalogue number: 3309 | Concentration: 0.1–100 µM |
| Chemical compound, drug | Rosuvastatin calcium | Tocris | Catalogue number: 6343 | Concentration: 0.1–100 µM |
| Chemical compound, drug | NB-598 | MedChem Express | Catalogue number: HY-16343 | Concentration: 0.1–100 µM |
| Chemical compound, drug | FGTI-2734 | MedChem Express | Catalogue number: HY-128350 | Concentration: 10 µM |
| Chemical compound, drug | FTI-277 hydrochloride | MedChem Express | Catalogue number: HY-15872A | Concentration: 10–30 µM |
| Chemical compound, drug | GGTI-289 trifluoroacetate | MedChem Express | Catalogue number: HY-15871 | Concentration: 10–30 µM |
| Chemical compound, drug | PD18352 | Tocris | Catalogue number: 4237 | Concentration: 2 µM |
| Chemical compound, drug | VX745 | Tocris | Catalogue number: 3915 | Concentration: 1 µM |
| Chemical compound, drug | Rapamycin | Merck | Catalogue number: R8781 | Concentration: 20 nM |
| Commercial assay, kit | CellTrace Violet Cell Proliferation Kit | Invitrogen | Catalogue number: C34557 | Concentration: 2.5 µM |
| Commercial assay, kit | MitoTracker Red FM | Invitrogen | Catalogue number: M22425 | Concentration: 100 nM |
| Software, algorithm | FlowJo software | BD Biosciences, developed by Treestar | RRID:SCR_008520 | Version 10.10.0 and above |
| Software, algorithm | Perseus | https://www.maxquant.org/perseus, PMID:27348712 | RRID:SCR_015753 | Version 1.6.6.0 |
| Software, algorithm | Spectronaut | Biognosys | Version 19 | |
| Software, algorithm | Prism | GraphPad | RRID:SCR_002798 | Version 9 or 10 |
| Other | Streptavidin Microbeads | Miltenyi Biotech | Catalogue number: 130-042-901 | Dilution: (1:10) |
Additional files
-
Supplementary file 1
Excel file containing terms for protein enrichment too large to fit in a PDF.
Related to Figure 1H, Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/109093/elife-109093-supp1-v1.xlsx
-
Supplementary file 2
Serum tests related to Figure 5.
- https://cdn.elifesciences.org/articles/109093/elife-109093-supp2-v1.docx
-
Supplementary file 3
Excel file containing proteomic data set (Naïve vs LPS +IL-4).
- https://cdn.elifesciences.org/articles/109093/elife-109093-supp3-v1.xlsx
-
Supplementary file 4
Excel file containing proteomic data set (Naïve vs LPS +IL-4 (Normal/CF +/-Fluvastatin)).
- https://cdn.elifesciences.org/articles/109093/elife-109093-supp4-v1.xlsx
-
Supplementary file 5
Statistics related to all figures.
- https://cdn.elifesciences.org/articles/109093/elife-109093-supp5-v1.docx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/109093/elife-109093-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
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Lipopolysaccharide stimulates dynamic changes in B cell metabolism to promote proliferation
eLife 14:RP109093.
https://doi.org/10.7554/eLife.109093.3
