Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: SLC7A5 regulates B cell metabolism and plasma cell differentiation independent of leucine transport

doi: 10.1093/jimmun/vkaf328

Figure Lengend Snippet: (A) Levels of phospho-mTOR (p-mTOR) in g Slc7a5 and gCtrl-transduced B cells after co-culture with 40LB cells for 24 hours. Representative flow cytometry plots (left) and mean fluorescence intensity (MFI) of p-mTOR normalized to the average of the gCtrl B cells in each experimental batch (right). B cells were stimulated with αRP105 for 2 days, transduced and restimulated with CpG for 24h. Cells were gated on FSC hi SSC lo NGFR + B cells as shown in Fig. S5A , where NGFR is a reporter for transduced cells. (B) S6 phosphorylation in g Slc7a5 or gCtrl-transduced B cells after co-culture with 40LB cells for 24h. Representative flow cytometry plots (left) and quantification of relative frequencies of phospho-S6 + B cells (right). Frequencies were normalized to the average of the gCtrl samples (indicated by the dotted line) in each experimental batch. Cells were gated on FSC hi SSC lo NGFR + B cells. (C) Schematic illustration of lysosome- and PI3K/AKT-dependent regulation of mTORC1 activity. (D-H) Extracellular flux (XF) analysis of extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) in g Slc7a5 and gCtrl-transduced B cells. B cells were stimulated with anti-RP105 for 3 days and re-stimulated with CpG for 1 day. (D) Representative time-series plot of ECAR at baseline and after challenge with 1 μM oligomycin (dotted line). (E) Quantification of baseline ECAR (left) and maximum ECAR (right). (F) Representative time-series plot of OCR at baseline and after sequential addition of 1 μM oligomycin, 1.5 μM FCCP, and 0.5 μM Rotenone/Antimycin A (each indicated by dotted lines). (G) Quantification of baseline OCR (left) and maximum OCR following addition of FCCP (right). (H) Plot of baseline OCR vs. ECAR. (I) PC differentiation of gCtrl transduced B cells and g Slc7a5 -transduced B cells expressing empty vector (−), myrAKT or RHEB Q64L . Representative flow cytometry plots (left) and quantification of CD138 + TACI + PC frequencies (right). Gating strategies for myrAKT and RHEB Q64L conditions are shown in Fig. S5C . (J) Analysis of phospho-S6 + gCtrl and g Slc7a5 -transduced B cells with or without ectopic expression of myrAKT or RHEB Q64L after 1 day co-culture with 40LB feeder cells. Representative flow cytometry plots (left) and quantification of p-S6 + cell frequencies (right). Data in A, B, D–J are from 6 mice and at least 2 independent experiments. Each dot represents a biological replicate. Shown are the mean ± SEM. Statistical significance was tested by two-tailed Student’s t test (A, I, J), paired t-test (E, G) with the following significance levels: * P < 0.05, ** P < 0.01, *** P < 0.001. For data in I and J, all experimental comparisons were prespecified; ANOVA was performed to confirm global significance, followed by hypothesis-driven individual comparisons tested using two-tailed Student’s t test.

Article Snippet: Expression constructs used in this study include: myrAKT (Addgene, 10841) ( 64 ) and RHEB Q64L (Addgene, 21050) ( 65 ).

Techniques: Activity Assay, Co-Culture Assay, Flow Cytometry, Fluorescence, Phospho-proteomics, Expressing, Plasmid Preparation, Two Tailed Test