MAZR is not essential for the differentiation of BM-derived mast cells.

<div><p>(A) Diagram shows qRTPCR analysis of <i>Mazr</i> expression in thymus, CD4⁺ and CD8⁺ T cells and in IgE-primed BM-derived mast cells (BMMC). Expression levels are normalized to <i>Hprt</i> expression and levels in thymocytes were set as 1 (100%). Columns represent a summary of three independent samples. Mean ± SEM is shown.</p> <p>(B) Histograms depict expression of cell surface markers on <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC (after 5 weeks of culture). Filled gray areas are isotype control stainings. Data are representative of three independent experiments.</p> <p>(C) Flow cytometric analysis showing up-regulation of FcεRI levels in <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC. Filled gray areas are isotype control stainings. The solid black line shows the levels of cell-surface bound IgE after 15 min incubation. The dotted line shows the levels of cell-surface bound IgE after overnight priming. Data are representative of three independent experiments. </p> <p>(D) Toluidine blue staining of 5 week-cultured <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC prepared by a cytospin. Magnification 20x.</p></div

Minor defects in early and late mast cell effector functions in the absence of MAZR.

<div><p>(A) Diagram shows qRTPCR analysis of <i>Mazr</i> expression in resting anti-TNP IgE-primed BMMCs and in BMMCs activated for the indicated time points with TNP. Expression levels are normalized to <i>Hprt</i> expression and levels in IgE-primed non-activated mast cells were set as 1 (100%). Data show summary of three samples analyzed. Mean ± SEM is shown.</p> <p>(B) Plasma histamine levels in a systemic anaphylaxis model are shown. <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice were primed (i.v.) with anti-TNP IgE, challenged 24 hours later by i.v. injection of TNP or PBS. Serum was collected 2 minutes post-injection and histamine levels were determined by ELISA, n=7.</p> <p>(C) Absorbance (OD) of Evans Blue dye extravasated in a passive cutaneous anaphylaxis model from the ears of <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice is shown. Mice were injected with PBS and anti-TNP IgE into left and right ear, respectively, and 24 hours later mice were challenged by i.v. injection with TNP/Evans Blue dye. Extravasation of Evans Blue dye in the ear was measured 4 hours later. Diagram shows summary of 9 mice. Mean ± SEM is shown.</p> <p>(D) Anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs were activated for 10 min with TNP or PMA/ionomycin. β-Hexosaminidase release levels of <i>Mazr</i>^<i>F/F</i> BMMCs were set to 1 (n=10).</p> <p>(E) Anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs were activated for 60 min with TNP. LTB₄ levels were determined by ELISA. Mean ± SEM is shown. (n=4).</p> <p>(F) Flow cytometric analysis of calcium flux in anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs that have been activated with TNP. Data are representative of 3 independent experiments. </p> <p>(G) Cytokine production of anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs that were activated by plate-bound TNP for 24 hours (at least 5 independent mast cell batches were analyzed). Cytokine production of <i>Mazr</i>^<i>F/F</i> BMMCs was set to 1.</p></div

Reduced mast cell numbers <i>in vitro</i> but normal mast cell homeostasis <i>in vivo</i>.

<div><p>(A) Diagram showing the cumulative numbers of c-kit⁺FcεRI⁺<i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC over the course of 5 weeks of culture. Cells were counted by CASY counter, then percentages of c-kit⁺FcεRI⁺ BMMC among PI-negative cells (= alive) was determined by flow cytometry. The summary of three independent experiments with a total of 6 independent cell batches is shown. Mean ± SEM is shown.</p> <p>(B) Number of PI-negative (= alive) <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC during 5 days of IL-3 starvation. The summary of 3 experiments is shown. Mean ± SEM is shown.</p> <p>(C) Toluidine blue staining of paraffin-embedded 5 µm ear sections of <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice showing mast cells in pink/purple color (examples indicated by arrowheads). Diagram at the right indicates mean ± SEM of mast cell number per field of view (fov) calculated over 10 individual sections per ear (n=4). Magnification 20x.</p> <p>(D) Percentage of EYFP⁺c-kit⁺FcεRI⁺ mast cells from peritoneal lavage of wild-type (Mazr^F/+Rosa26^+/EYFPMcpt5Cre) and mast cell-specific MAZR-null (Mazr^F/FRosa26^+/EYFPMcpt5Cre) mice (n=8 and 9, respectively).</p></div

Gene expression analysis of non-activated MAZR-deficient BMMCs.

<div><p>(A) Gene expression profiles from IgE-primed (but non-activated) <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC were determined using Agilent arrays. Data were analyzed using GeneSpring software as described in materials and methods. The scatter plot indicates the 128 genes that are dysregulated in the absence of MAZR (log₂ expression levels; ≥2 fold-difference, P≤0.1). Numbers at the upper-left or lower-right corners show the number of genes specifically expressed in <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> (Y-axis) and <i>Mazr</i>^F/F (X-axis) BMMCs. The highlighted genes are selected from the top-ten hits with the largest-fold difference between <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC.</p> <p>(B) qRTPCR analysis of genes selected from the microarray experiments. Graphs represent relative expression levels of genes up- and down-regulated in the absence of MAZR (normalized to <i>Hprt</i>). Expression levels in <i>Mazr</i>^F/F samples were set to 1. Mean ± SEM is shown. Data are means of the results from duplicated qRTPCRs of one batch of mast cells. The IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC samples were from a different batch compared to the ones used for probing with Agilent arrays.</p></div