Supplementary Figure 1. Xbp1 deficiency does not alter hematopoietic cellularity.

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1 Supplementary Figure 1 Xbp1 deficiency does not alter hematopoietic cellularity. Absolute number of leukocytes obtained from bone marrow (BM, 2 tibias and 2 femurs per mouse) of Xbp1 f/f and Xbp1 Vav1 mice (n = 3 per genotype). Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). NS, not significant (P > 0.05, Student s t-test). Data are representative of at least three independent experiments with at least three mice per genotype per experiment.

2 Supplementary Figure 2 Xbp1 deficiency minimally influences most leukocyte subsets. (a) Flow cytometry of splenic CD3 + T cells and B220 + B cells in Xbp1 f/f and Xbp1 Vav1 mice. Numbers adjacent to outlined areas indicate percent CD3 + B220 T cells (top left) or CD3 B220 + B cells (bottom right). (b,c) Frequency of T cells (b) and B cells (c) in spleens of Xbp1 f/f and Xbp1 Vav1 mice (n = 3 per genotype). (d) Flow cytometry of splenic FcεRI + basophils in Xbp1 f/f and Xbp1 Vav1 mice. Numbers adjacent to outlined areas indicate percent SSC low FcεRI + basophils gated on non-b and non-t cells. (e) Splenic basophil frequency in Xbp1 f/f and Xbp1 Vav1 mice (n = 3 per genotype). (f) Flow cytometry of splenic CD11b + Ly6G + neutrophils in Xbp1 f/f and Xbp1 Vav1 mice. Numbers adjacent to outlined areas indicate percent CD11b + Ly6G + neutrophils. (g) Splenic neutrophil frequency in Xbp1 f/f and Xbp1 Vav1 mice (n = 3 per genotype). (h) Flow cytometry of splenic CD11b + F4/80 + macrophages in Xbp1 f/f and Xbp1 Vav1 mice. Numbers adjacent to outlined areas indicate percent CD11b + F4/80 + macrophages. (i) Splenic macrophage frequency in Xbp1 f/f and Xbp1 Vav1 mice (n = 3 per genotype). (j) Flow cytometry of splenic CD11c + dendritic cells (DCs) in Xbp1 f/f and Xbp1 Vav1 mice. Numbers adjacent to outlined areas indicate percent B220 CD11c + macrophages. (k) Splenic DC frequency in Xbp1 f/f and Xbp1 Vav1 mice (n = 3 per genotype). Each symbol (b,c,e,g,i,k) represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). NS, not significant (P > 0.05); *P < 0.05 (Student s t-test). Data are representative of at least three independent experiments with at least three mice per group in each.

3 Supplementary Figure 3 The Vav1-Cre transgene does not affect eosinophil differentiation. Flow cytometry of CCR3 + Siglec-F + eosinophils from the blood of Xbp1 f/f, Xbp1 WT/WT Vav1-Cre +, and Xbp1 Vav1 mice (n = 2 mice per group). Numbers adjacent to outlined areas indicate percent CCR3 + Siglec-F + eosinophils. Data are representative of at least 2 independent experiments with at least two mice per group in each.

4 Supplementary Figure 4 Cre expressed from the Vav1-Cre transgene results in efficient deletion of Xbp1 in basophils, neutrophils and GMPs. (a,b,c) Quantitative PCR analysis of the floxed Xbp1 exon 2 in bone marrow basophils (a), neutrophils (b), and GMPs (c) sorted from Xbp1 f/f and Xbp1 Vav1 mice (n = 3 mice per genotype). Results were normalized to those of Actb and are presented relative to those of Xbp1 f/f cells. *P < 0.05 and **P < 0.01 (Student s t-test). Data are from one experiment (a,b; mean and s.e.m.) or are representative of more than three experiments with three mice per genotype (c; mean and s.e.m.).

5 Supplementary Figure 5 Inhibition of IRE1α does not affect the survival of mature eosinophils. (a) Flow cytometry of annexin-v and propidium iodide (PI) staining of Siglec-F + SSC hi bone marrow cells after 24 h in vitro incubation with vehicle dimethyl sulfoxide (DMSO) or 20 μm of the IRE1α inhibitor 4μ8C. Numbers adjacent to outlined areas indicate percent PI + Annexin-V + late apoptotic eosinophils (top right), PI Annexin-V + early apoptotic eosinophils (bottom right), or PI Annexin-V live eosinophils. (b) Frequency of eosinophils (eos) of total bone marrow cells after 24 h in vitro incubation with DMSO or 20 μm 4μ8C (n = 3 cultures per condition). (c) Percent viability of eosinophils from bone marrow cultures after 24 h in vitro incubation with DMSO or 20 μm 4μ8C (n = 3 cultures per condition). Each symbol (b,c) represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). NS, not significant (P > 0.05, Student s t-test). Data are representative of two independent experiments with three independent biological replicates per condition.

6 Supplementary Figure 6 Overexpression of GATA-2 in GMPs drives commitment to the eosinophil lineage. (a) Microscopy of GMPs transduced with GFP control (GFP-RV, Control ) or GATA-2 expressing (GATA-2-RV, GATA-2 TD ) retroviruses after indicated timepoints in culture. Scale bars, 5 μm. (b) Quantitative PCR analysis of Prg2 and Epx in GMPs transduced as in a after 96 h in culture; results were normalized to those of Actb (n = 4 cultures per virus). (c) Microscopy of GATA-2-transduced Xbp1 f/f GMPs and GATA-2-transduced Xbp1 Vav1 GMPs after 96 h in culture. Scale bars, 5 μm. ***P < (Student s t-test). Data are representative of two independent experiments with at least three independent biological replicates per condition (a c; mean and s.e.m. in b).