l, m Plasma concentrations of FFAs ( l ), AST and ALT ( m ). j Relative mRNA expression of genes involved in fibrogenesis ( n = 3). i Representative IHC images for alpha-Sma, collagen I, desmin, and Sirius red staining of liver tissues. h Relative mRNA expression of genes involved in cell death pathways (n = 3). Quantification of TUNEL-positive cells (bottom panel). g TUNEL staining of the liver (top panel). Quantification of ROS formation by measuring DHE-positive cells in liver tissues (right panel). f Representative images of DHE-stained cells (left panel). d Relative mRNA expression levels of genes involved in pro-inflammation ( n = 3). Percentage of F4/80 + Cd11b + cells ( n = 3) (right panel). b Flow cytometry analyses of the expression of F4/80 and Cd11b (left panel). Quantification of hCLS formation and F4/80- and Cd11b-positive areas (right panel). a Representative IHC images of F4/80- and Cd11b-stained liver tissues (left panel). a - m Cxxc5 +/+ and Cxxc5 -/- mice were fed a HFD for 12 weeks and injected with CCl 4 twice a week for the final 4 weeks to induce NASH ( n = 7 per group). (I) Flow cytometry of the proportion of macrophages (F4/80 + CD11B + ), monocytes (LY6C + CD11B + ), and T cells (CD3 + ) in CD45 + immune cells among three groups (n = 3-4, per group).ĭeletion of Cxxc5 ameliorates NASH progression. (H) Immunostaining for CD45 (red), CD68 (green), and overlays with DAPI-labeled nuclei (blue) showing macrophages in the mouse aorta (scale bars: 100 mum). (G) Immunostaining for vimentin (white) and DAPI overlay in the thoracic and abdominal aorta form mice subjected to 4 weeks of Ang II induction (scale bars: 500 mum). (F) Immunostaining for calponin (red), alphaSMA (green), and overlays with DAPI-labeled nuclei (blue) showing smooth muscle cells in the mouse aortic media (scale bars: 100 mum). (D,E) Immunofluorescence analysis for vimentin (white) (D) and CD45 (purple) (E) and overlays with DAPI-labeled nuclei (blue) showing fibroblasts and immune cells in the human aorta (scale bars: 100 mum). Lu means the lumen side of the vascular wall, and A means the adventitia side of the vascular wall. (C) Immunofluorescence analysis for SMMHC (red), alphaSMA (green), and overlays with DAPI-labeled nuclei (blue) showing smooth muscle cells in the human vascular media (scale bars: 100 mum). the corresponding normal aorta of humans (A) and mice (B). (A,B) Fractions of major cells types in aneurysmal aorta vs. ( G ) Quantification of the percentage of TIM3 + cells ( n = 6-8).įIGURE 2 Alteration of cellular composition in the diseased status of mice and humans. ( E - G ) Representative FACS analysis of 2 independent experiments of macrophages (CD11b + F4/80 + TIM3 + ) out of all CD45 + cells in the 8505c xenograft tumors ( E ) and C643 xenograft tumors ( F ). Representative immunofluorescence images of 3 independent experiments. ( B - D ) 20X and 63X images of 5 um thick 8505C xenograft tumor sections immunostained with TIM3-PE antibody (red). Representative results of 3 independent experiments. TIM3 mRNA expression was normalized by beta-actin. The lower panel represents the expression of actin. Lane 1, RAW264.7 cells lanes 2-4, 8505C xenograft tumors ( n = 3) lanes 5-7, C643 xenograft tumors ( n = 3). Total RNA was isolated, and the mRNA expression of TIM3 was examined by RT-PCR analysis. Upper panel: TIM3 mRNA expression in RAW264.7 cells and in ATC xenografts. Figure 4 TIM3-positive macrophages were recruited to ATC xenograft tumors.
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