Supplementary Figure 5

ER stress induced death of cDC1 is CHOP and JNK independent (a) transmission electron micrographs of FACS-sorted cDC1s and cDC2s derived from the lung or intestine of XBP1ΔDC or XBP1^fl/fl mice. The full arrow indicates ER with normal appearance. The dashed line indicates an ER aggregate. Scale bars, 1 μm. (b) immunofluorescence of cDC2s, sorted by flow cytometry from lung and LP-SI of Xbp1^fl/fl and XBP1ΔDC mice, and stained with an anti-KDEL (ER-marker) antibody. Nuclei were stained with Hoechst. Scale bar: 4,43 μm. (c) RT-qPCR analysis of Ddit3 mRNA among total RNA prepared from in-vitro cultured DCs. BM cells of Xbp1^fl/fl, XBP1ΔDC or XBP1ΔDC-CHOP^nil mice were cultured with GM-CSF and treated with tunicamycin (TM) or vehicle. Results are normalized to gapdh and ywhaz. Bar graph represents 1 sample. (d) immunoblot analysis of phospho-JNK in flow cytometry sorted splenic cDC1s of XBP1fl/fl or XBP1ΔDC mice. Total JNK levels serve as loading control. Each lane represents one mouse. ^∗ indicates an aspecific band. (e) immunoblot analysis of phospho-JNK of splenic XBP1ΔDC CD11c⁺ cells of XBP1ΔDC mice treated with CC-930 or vehicle. Total JNK and β-tubulin levels serve as loading control. Each lane represents one mouse. (f) scheme illustrating activation of ATF6 and PERK branches ensuing XBP1 deletion in cDC1s. ATF6 and PERK activity resulted in enhanced transcription of target genes of both pathways. Loss of XBP1 also results into hyperactivation of IRE1 kinase activity and JNK phosphorylation. Nor CHOP (encoded by the Ddit3 gene) nor JNK played a critical role in mediating cell death of XBP1Δ cDC1s in the lung.