Extended Data Fig. 1
From: Blockade of IL-6 signaling alleviates atherosclerosis in Tet2-deficient clonal hematopoiesis
a. Ldlr−/− male mice were transplanted with WT or Tet2KO male mice bone marrow; after 6 weeks reconstitution, WT→Ldlr−/− (n = 9) and Tet2KO→Ldlr−/− (n = 10) mice were fed a western diet for 12 weeks. Bar graph shows total lesion area in the aortic root. Two-tailed unpaired t-test. b-c. WT and Tet2KO macrophage were pretreated with or without IL-1β (100 μg/ml) blocking antibodies for 1 hr, then treated with LPS (20 ng/ml) for 4 hours followed by nigericin (5 μg/ml) for another 30 mins. Secreted IL-1β and IL-6 were determined by ELISA. n = 5 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. d. Bone marrow derived macrophages were pre-incubated with IL-6R blocking antibodies (100 μg/ml) for 1 hour and then challenged with or without IL-6 (25 ng/ml). qPCR was used to measure the mRNA level of Socs3 expression. n = 3 independent experiments. One-way ANOVA followed by Tukey’s multiple-comparison test. e-g. Peripheral blood white blood cells counts, RBC counts and platelet counts after 12 weeks western diet. n = 15 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. h-l. Body weight, plasma cholesterol and triglycerides, liver/body weight ratio, spleen/body weight ratio after 12 weeks western diet. m. Immunoblot of isolated splenic monocytes and neutrophil Caspase1, cleaved IL-1β and β-actin after 12 weeks western diet. n = 6 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test or two-tailed unpaired t-test. n. Immunofluorescence staining of macrophage (anti-Mac2, Green), TREM2 (Red) in aortic roots and quantification of TREM2+Mac2+ cells as the percentage of total Mac2+ cells per lesion area in aortic root cross-sections. Scale Bar, 50μm. n = 15 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. o. Immunofluorescence staining of macrophage (anti-Mac2, Green), IL-1β expression (Red) in aortic roots and quantification of IL-1β mean fluorescence intensity per lesion area in aortic root cross-sections. Scale Bar, 50μm. n = 15 mice per group. One-way ANOVA followed by Tukey’s multiple-comparison test. p. Quantification of CSF1R expression in monocytes of Ctrl and Tet2+/− mice. n = 5 mice in ctrl group. n = 6 mice in Tet2 + /- group. Two-tailed unpaired t-test. All the Data are Mean ± SEM.
