Supplementary Figure 6: Disruption of Mcpt8 expression levels in Basoph8 mice does not impact the outcome of CLP of moderate severity.

(a, b) Mcpt8 expression levels are significantly reduced or undetectable in basophils from mice with one or two Basoph8 gene copies, respectively. Mcpt8 mRNA (a) and protein (b) expression levels in basophils that were sorted from bone marrow cells harvested from naïve mice (a) or from FcεRIα⁺ CD49b⁺ c-Kit^– bone marrow-derived cells cultured in the presence of IL-3 (b). The qPCR data in a and western blot analysis in b are representative of 2 independent experiments performed, each of which gave similar results. The Western blot analysis in b was cropped from the original blot shown in Supplementary Fig. 7. (c, d) Basophil-derived TNF-deficient mice with one Basoph8 gene copy (mice with a significant reduction in Mcpt8 expression levels) exhibit similar mortality and morbidity as basophil-derived TNF-deficient mice with two Basoph8 gene copies (mice with a total Mcpt8 deficiency). Survival after moderate CLP in littermate control mice (Tnf^fl/fl = Basoph8 ^-/- x Tnf^fl/fl, n = 15), Basoph8^+/- x Tnf^fl/fl (n = 7) and Basoph8^+/+ x Tnf^fl/fl (n = 7). *P < 0.05 and **P < 0.01 vs. Tnf^fl/fl mice. P values by Logrank (Mantel-Cox test). (d) Drop in body temperature in Tnf^fl/fl mice (n = 7), Basoph8^± x Tnf^fl/fl mice (n = 13) and Basoph8^+/+ x Tnf^fl/fl mice (n = 17) at 24 h after CLP. P values by Mann-Whitney test. Data in c were pooled from the n = 3 independent experiments performed, each of which gave similar results. Data in d are shown as means + SEM with circles representing values for individual mice and were pooled from n = 3 independent experiments performed, each of which gave similar results. (e-j) Mice with one Basoph8 gene copy (that is, Basoph8^± x Tnf ^+/+ mice with no Mcpt8 protein expression, but which express basophil-derived TNF) exhibit morbidity rates similar to their littermate controls [Tnf ^+/+ mice]). (e-j) Drop in body temperature (e), colony forming units (CFU) in peritoneal cavity (f) and blood (g), amount of intraperitoneal TNF (h), and numbers of neutrophils (i) and macrophages (j) in the peritoneal live-cell population analyzed by flow cytometry (Gr-1⁺ CD11b⁺ cells for neutrophils and F4/80⁺ CD11b⁺ for macrophages) in Basoph8^-/- x Tnf^+/+ mice and Basoph8^± x Tnf^+/+ mice at 24 h after CLP. Data are shown as means + SEM with circles representing values for individual mice (n = 23 in e, n = 19 in f, n = 16 in g, n = 14 in h and n = 7 in i and j for Basoph8^-/- x Tnf^+/+ mice; and n = 9 in e, n = 14 in f, n = 13 in g, n = 7 in h and n = 5 in i and j for Basoph8^± x Tnf^+/+ mice. Data were pooled from n = 3 independent experiments performed, each of which gave similar results.