Fig. 4.

Sec22b is essential for ER expansion and structure in PCs. (A) Expression of Ern1 (encoding Ire1a), Atf4, Atf6, and Ddit3 (encoding Chop) in fragments per kilobase of exon per million reads mapped (FPKM) determined by RNAseq in Sec22b^WT and Sec22b^B-KO PCs. (B) Heatmap showing the relative expression of selected ER stress genes from PCs generated from Sec22b^WT and Sec22b^B-KO splenocytes after 2 d of LPS stimulation, determined by Biomark multiplex qPCRs at steady state. (C) Flow cytometric quantification of ER-Tracker MFI (geometrical mean) on PCs generated from Sec22b^WT and Sec22b^B-KO splenocytes after 2 or 4 d of LPS stimulation. n = 4, one representative experiment shown out of 3. (D) Confocal microscopy images of Sec22b^WT and Sec22b^B-KO PCs obtained from splenocytes stimulated with LPS for 2 d. Cells were stained with an anti-IgM antibody to detect intracellular IgM, an anti-calnexin antibody to detect the ER and an anti-IRF4 antibody. Nuclei were counterstained with Hoechst. Images are representative of 60 Sec22b^WT PC and 71 Sec22b^B-KO PC from two mice per group. (E) Electron microscopy images of Sec22b^WT (Left) and Sec22b^B-KO (Right) PCs obtained from splenocytes stimulated with LPS for 2 d (Top) or 4 d (Bottom). Red arrowheads indicate ER sheets. (Scale bar, 1 mm or 0.5 mm.) Images are representative of 64 WT PCs (9 on day 2 and 55 on day 4) and 39 KO PCs (21 on day 2 and 18 on day 4). Data are from five mice per genotype out of two experiments. The heatmaps were generated using the heatmapper.ca website, row Z score based on (2-^ΔCt) values. The P-values were determined with the two-tailed Mann–Whitney nonparametric test *P < 0.05. Sec22b^WT controls were mb1cre⁺ and Sec22b^fl/fl/Sec22b^fl/+ mice.