Journal: Nature Communications

Article Title: mTORC1 coordinates an immediate unfolded protein response-related transcriptome in activated B cells preceding antibody secretion

doi: 10.1038/s41467-019-14032-1

Figure Lengend Snippet: a C57BL/6 mouse splenocytes were stained for intracellular Xbp1s. Gating criteria are shown in Supplementary Fig. . Shown is a representative histogram and MFI of three animals, which are summarized as the mean (line), 75% CI (box), and 95% CI (whisker) and individual points. One-way ANOVA with Tukey post-test was performed to generate p values. b Intracellular phosphorylated S6 protein staining was performed on follicular (FO) and MZ B cells from mice treated with rapamycin or vehicle control every other day for a total of four treatments. Counts as percent of maximum are displayed with quantification on right. p value is two-tailed Student’s t -test. Summary data for all animals presented as individual data points, mean and SEM. c – g RNA-seq was performed on splenic follicular and MZ B cells and short-lived (B220 + ) and long-lived (B220 − ) BM PCs from five individual B6.Blimp +/GFP adults. c Differentially expressed genes were averaged by group and gene co-regulation was determined by hierarchical clustering by Pearson correlation with a grouping cutoff ( k ) of six chosen using best of 26 indices by NbClust . Gene expression is averaged by group ( n = 5) for clarity and displayed as z score across each row. d Gene ontology clustering enrichment analysis of selected co-expression clusters is shown. Indicated is the founder term for each GO term cluster followed by gene numbers for that term. Bar length indicates the enrichment score for the GO cluster. e Volcano plots showing genes differentially expressed in MZ B cells over follicular B cells for all genes (first panel), UPR hallmark genes, mTORC1-signaling hallmark genes, the top 250 upregulated genes in B220 + BM PCs versus follicular B cells and the top 250 genes upregulated in B220 − BM PCs versus follicular B cells. Genes are color coded by adjusted p -value (red = p < 0.05). Adjusted p -value is BH-adjusted (eBayes method—Limma). f Log 2 TPM expression magnitude of selected genes is shown as mean (line), 75%CI (box) and 95%CI (whisker) as well as individual data points (jitter). (* p < 0.05, ** p < 0.01, *** p < 0.001; differential expression statistic: BH-adjusted p -value—eBayes method—Limma). g GSEA was performed using the BROAD UPR and mTORC1-signaling hallmark gene sets as well as B220 + and B220 − upregulated gene sets described in e comparing MZ B and follicular B cells. FDR-q values computed using 1000 geneset permutations. Source data are provided as a Source Data file.

Article Snippet: ATF-4 (D4B8)[1:1000], XBP1s (E8Y5F) [1:1000], p58IPK (C56E7)[1:1000], ATF-6 (D4Z8V) [1:1000], BiP (C50B12)-PE[1:200], pAKT (S473) (D9E)-PE[1:100] and pS6(S235/236) (D57.2.2E)-PE-Cy7[1:200] were purchased from Cell Signaling Technology.

Techniques: Staining, Whisker Assay, Control, Two Tailed Test, RNA Sequencing, Gene Expression, Expressing, Quantitative Proteomics

Journal: Nature Communications

Article Title: mTORC1 coordinates an immediate unfolded protein response-related transcriptome in activated B cells preceding antibody secretion

doi: 10.1038/s41467-019-14032-1

Figure Lengend Snippet: a The conventional model for ER remodeling during plasma cell differentiation holds that Blimp1-mediated increases in immunoglobulin expression causes the chaperone BiP to release from the luminal tails of IRE1α, allowing it to dimerize and mediate Xbp1 mRNA splicing. In this model Xbp1s is the main effector of ER remodeling. Paradoxically, with this scenario activation of PERK and ATF6 would occur by the same mechanism, causing translation inhibition and increased chance of apoptosis downstream of PERK activation. b Revised model wherein, preceding Blimp1 induction, mTORC1 signaling facilitates the upregulation of plasma cell-affiliated UPR genes, including chaperones, ERAD machinery, and enzymes necessary for enhancing protein secretion. The resulting “primed” ER is then better equipped to handle increased protein load following Blimp1 induction. At this point the UPR is tuned so that we see activation of the Xbp1-splicing activity of IRE1α without RIDD activity and activation of PERK.

Article Snippet: ATF-4 (D4B8)[1:1000], XBP1s (E8Y5F) [1:1000], p58IPK (C56E7)[1:1000], ATF-6 (D4Z8V) [1:1000], BiP (C50B12)-PE[1:200], pAKT (S473) (D9E)-PE[1:100] and pS6(S235/236) (D57.2.2E)-PE-Cy7[1:200] were purchased from Cell Signaling Technology.

Techniques: Clinical Proteomics, Cell Differentiation, Expressing, Activation Assay, Inhibition, Activity Assay