Journal: The Journal of Clinical Investigation

Article Title: Erythroid lineage Jak2 V617F expression promotes atherosclerosis through erythrophagocytosis and macrophage ferroptosis

doi: 10.1172/JCI155724

Figure Lengend Snippet: Control and VFEpoR mice were fed a Western diet and treated with LDLR ASO and EPO (3 times per week) for 12 weeks. ( A ) H&E staining of aortic root sections and quantification of absolute lesion and necrotic core area. Necrotic core regions indicated by broken lines. Scale bar: 200 μm. ( B ) Aortic root sections were stained with Masson’s trichrome staining for fibrous cap (red, outlined by broken lines) and collagen (blue) content area, and then quantified as the ratio of total lesion area. Scale bar: 100 μm. ( C ) Iron (Perl’s blue) and redox-active iron deposition (Perl’s blue + DAB), IHC staining of RBCs (anti-Ter119) and macrophages (anti-Mac2) in aortic roots. Bar graph shows quantification of iron (II + III)–positive and erythrophagocytosis (Ter119 + Mac2 + ) cell counts per section. Scale bar: 100 μm. ( D ) TUNEL and immunofluorescence staining of macrophage (anti-Mac2) in aortic roots and quantification of TUNEL-positive cell counts per section. Scale bar: 50 μm. ( E ) Lipid peroxidation product 4-HNE staining, quantified as the percentage of total lesion area. Scale bar: 100 μm. ( F ) Immunofluorescence staining of TfR1 and macrophage (anti-Mac2), and quantification of TfR1 and macrophage costaining cell counts per section. Scale bar: 50 μm. ( G ) Aortic root sections were immunostained for Mac2 and quantified as absolute Mac2-positive area. Scale bar: 250 μm. ( H ) Lesions were stained for citrullinated histones (H3Cit) and activated neutrophils using myeloperoxidase (MPO); the overlap H3cit and MPO (NETs) cell counts were quantified. Scale bar: 50 μm. Unpaired 2-tailed t test or Mann-Whitney U test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Identification of macrophages, NETosis, proliferation, and endothelial cells in atherosclerotic lesions were performed by immunostaining using anti-Mac-2 (Cedarlane, CL8942AP, 1:10,000), biotinylated myeloperoxidase (MPO) (R&D Systems, BAF3667, 1:30), anti-histone H3 antibody (Abcam, ab5103, 1:300), anti-Ki67(Abcam, ab15580, 1:100), anti-vWF (Abcam, ab117113, 1:100), anti–p-MLKL (Abcam, 196436, 1:100), and anti-TfR (Thermo Fisher Scientific, 13-6800, 1:100).

Techniques: Control, Western Blot, Staining, Immunohistochemistry, TUNEL Assay, Immunofluorescence, MANN-WHITNEY