Figure S4.
Differentially expressed genes, affected organelles, and IgM levels in in vitro–generated Jagn1-deficient plasmablasts.(A) Heatmap depicting relative expression levels of 521 dysregulated genes as assessed by QuantSeq 3′ mRNA sequencing of mRNA from Jagn1ΔB (right) versus control (left) sorted plasmablasts (data from four individual mice are shown; padj < 0.01). Color gradient from blue to red indicates increased expression levels (z-score). Genes in sterol and cell cycle categories are shown by blue lines on the left and are mostly down-regulated in cells from Jagn1ΔB mice. Genes involved in apoptosis and stress response/UPR are displayed by red lines on the left and are mostly up-regulated in cells from Jagn1ΔB mice. Genes described with the GO keyword ER are marked by gray lines on the left. (B) Functional enrichment analysis of down-regulated genes determined by QuantSeq 3′ mRNA sequencing of mRNA from Jagn1ΔB versus control plasmablasts (data from four individual mice) as assessed by DAVID GO analysis. Top GO terms enriched in Jagn1ΔB plasmablasts ranked by the negative logarithm of the false discovery rate (FDR) are shown (padj < 0.01). (C) Golgi Cytopainter staining in plasmablasts (CD22−CD138+) isolated from mice of the indicated genotypes after 4 d of LPS stimulation. Representative histogram overlays (left panel) and mean fluorescence intensities (MFI; right panel; n > 2) are shown. The dotted line in the histogram indicates the background control. Data are shown for one of two independent experiments. (D) LysoTracker staining in plasmablasts (CD22−CD138+) from mice of the indicated genotypes after 4 d of LPS stimulation. Representative histogram overlays (left panel) and mean fluorescence intensities (MFI; right panel; n = 4) are shown. The dotted line in the histogram indicates the background control. Data are shown for one of two independent experiments. (E) Immunoblot analysis for IgM heavy chains on lysates prepared from sorted plasmablasts from mice of the indicated genotypes, analyzed after 4 d of LPS stimulation. β-Actin protein levels are shown as loading controls. Data are shown for one of two independent experiments. (F) Immunoblot analyses to test for secreted IgM heavy and light chains from supernatants of cell cultures as in C. Data are shown for one of two independent experiments. (G) Ig concentrations in sera from mice of the indicated genotypes (n = 3) assessed with an isotype-multiplexing assay. Fold reductions are indicated above bar diagrams. Data are shown for one of two independent experiments. For panels C, D, and G, each data point represents an individual mouse. P values were calculated using the Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent means ± SD.

Differentially expressed genes, affected organelles, and IgM levels in in vitro–generated Jagn1-deficient plasmablasts. (A) Heatmap depicting relative expression levels of 521 dysregulated genes as assessed by QuantSeq 3′ mRNA sequencing of mRNA from Jagn1ΔB (right) versus control (left) sorted plasmablasts (data from four individual mice are shown; padj < 0.01). Color gradient from blue to red indicates increased expression levels (z-score). Genes in sterol and cell cycle categories are shown by blue lines on the left and are mostly down-regulated in cells from Jagn1ΔB mice. Genes involved in apoptosis and stress response/UPR are displayed by red lines on the left and are mostly up-regulated in cells from Jagn1ΔB mice. Genes described with the GO keyword ER are marked by gray lines on the left. (B) Functional enrichment analysis of down-regulated genes determined by QuantSeq 3′ mRNA sequencing of mRNA from Jagn1ΔB versus control plasmablasts (data from four individual mice) as assessed by DAVID GO analysis. Top GO terms enriched in Jagn1ΔB plasmablasts ranked by the negative logarithm of the false discovery rate (FDR) are shown (padj < 0.01). (C) Golgi Cytopainter staining in plasmablasts (CD22−CD138+) isolated from mice of the indicated genotypes after 4 d of LPS stimulation. Representative histogram overlays (left panel) and mean fluorescence intensities (MFI; right panel; n > 2) are shown. The dotted line in the histogram indicates the background control. Data are shown for one of two independent experiments. (D) LysoTracker staining in plasmablasts (CD22−CD138+) from mice of the indicated genotypes after 4 d of LPS stimulation. Representative histogram overlays (left panel) and mean fluorescence intensities (MFI; right panel; n = 4) are shown. The dotted line in the histogram indicates the background control. Data are shown for one of two independent experiments. (E) Immunoblot analysis for IgM heavy chains on lysates prepared from sorted plasmablasts from mice of the indicated genotypes, analyzed after 4 d of LPS stimulation. β-Actin protein levels are shown as loading controls. Data are shown for one of two independent experiments. (F) Immunoblot analyses to test for secreted IgM heavy and light chains from supernatants of cell cultures as in C. Data are shown for one of two independent experiments. (G) Ig concentrations in sera from mice of the indicated genotypes (n = 3) assessed with an isotype-multiplexing assay. Fold reductions are indicated above bar diagrams. Data are shown for one of two independent experiments. For panels C, D, and G, each data point represents an individual mouse. P values were calculated using the Student’s t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars represent means ± SD.

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