Additional file 3 of Serum meprin α levels for the detection of systemic inflammatory response syndrome

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Supplementary Material 3. Fig. S3 Characterization of LMα mice. (A) Scheme illustrates the genotype of transgenic mice designated as “control” and “LMα”. Both strains were bred on a C57BL7/6J background harboring a homozygous (+/+) insertion in the Rosa26 locus composed of a CAG-promotor (CAG), a loxP site (►)-flanked neomycin resistance (Neo)/STOP cassette, a cDNA sequence encoding HA-tagged murine meprin α (Mep1a-HA), an internal ribosomal entry site (IRES) and a cDNA sequence encoding enhanced green fluorescent protein (EGFP). LMα mice, but not control mice, also carry heterozygous (+/-) downstream of the Lyz2 promoter a substitution of the Lyz2 gene by a gene encoding cre recombinase. (B) Western blot detection of meprin α in the bone marrow of K5Mα (CreERT2+) and LMα (CreERT2+) as well as respective control (CreERT2-) mice. Gapdh was detected as reference. (C) Age of LMα (n=45) and respective control (n=44) mice in weeks. Violin plots display data distribution and mark median values (horizontal drawn-through line). (D) Hematocrit (HCT), (E) red blood cell count (RBC), (F) mean corpuscular volume (MCV), (G) red blood cell distribution width– coefficient of variation (RDW-CV), (H) hemoglobin concentration (HGB), (I) mean corpuscular hemoglobin (MCH), (J) mean corpuscular hemoglobin concentration (MCHC), (K) platelet count (PLT), (L) mean platelet volume (MPV), (M) white blood cell count (WBC), (N) neutrophil count, (O) lymphocyte count, (P) monocyte count, (Q) eosinophil count and (R) basophil count measured in EDTA-whole blood samples from control and LMα mice. Data points show respective values detected in individual control (n=44) and LMα (n=46) mice. Violin plots display data distribution and mark median values (horizontal drawn-through line). Reference ranges marked by black dotted lines and mean values are marked by black dashed lines. (S) Proportion of B cells (CD19+), T cells (CD3+ NK1.1-), neutrophils (CD11b+ Ly6G+), monocytes (CD11b+ Ly6G-, NK1.1-), NK cells (NK1.1+ CD3-) and NK T cells (NK1.1+ CD3+) within CD45+ leukocytes in the peripheral blood of control and LMα mice measured by flow cytometry. Data points show respective values detected in individual control (n=16-22) and K5Mα (n=20-22) mice. Bar charts and error bars display mean values and respective standard errors of the mean (SEM). (T) Proportion of CD11b+ Ly6G-, CD11b+ Ly6Glow and CD11b+ Ly6Ghigh CD45+ leukocytes in the bone marrow of control and LMα mice measured by flow cytometry. Data points show respective values detected in individual control (n=20) and K5Mα (n=22) mice. Bar charts and error bars display mean values and respective SEM. (U) Heatmap displays log2-transformed concentrations of interleukin-1 alpha (IL-1α), interleukin-6 (IL-6), interleukin-18 (IL-18), tumor necrosis factor alpha (TNFα), granulocyte colony stimulating factor (G-CSF), C-X-C motif chemokine ligand 1 (CXCL1) and CC motif chemokine ligand 2 (CCL2) in the serum of control (n = 28) and LMα mice (n = 30). Higher (red) and lower (blue) concentrations are highlighted by color gradients. Cytokine/chemokine levels were quantified by a custom-generated bead-based multiplex assay (LEGENDplex, Revvity). Statistically significant differences (P<0.05) were tested by two-tailed unpaired t-test and labeled by an asterisk (*). Graphical illustration in (A) was created with BioRender.com

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