Fig 1: The role of mitoNEET in attenuation of obesity-mediated arterial stiffness by pioglitazone. Expression of Cisd1 (coding for mitoNEET) in the a PVAT (n = 3, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively) and b aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). *P < 0.05, **P < 0.01, and ***P < 0.001. c Immunofluorescence staining for mitoNEET (CISD1, red) and DAPI (blue) in the thoracic aorta and PVAT. Each scale bar is 200 µm. d The correlation between PWV and genes in the thoracic aorta (n = 5, 3, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). Scatter plot illustrating the Spearman’s correlation of normalized reads per mouse between PWV and mitoNEET (Cisd1), CTSS (Ctss), and MMP-12 (Mmp12). Spearman’s rank correlation coefficients r and P value are provided in each plot. *P < 0.05. e ChIP assay in 3T3-L1 undifferentiated and Raw 264.7 cells treated with pioglitazone. The schematic representation shows the location of primers flanking putative PPAR?-binding sites (grey rectangles) in Cisd1. Sequence containing the potential PPAR?-binding sites in Cisd1 loci was amplified by real-time polymerase chain reaction in 3T3-L1 undifferentiated and Raw 264.7 cells. Data were collected from three independent experiments
Fig 2: Binding of PPAR? in Ctss and Mmp12. ChIP assay in Raw 264.7 and 3T3-L1 undifferentiated cells treated with pioglitazone. a, b The schematic representation shows the location of primers flanking putative PPAR?-binding sites (grey rectangles) in Ctss and Mmp12. Sequences containing the potential PPAR?-binding sites in Ctss and Mmp12 loci were amplified by real-time polymerase chain reaction in (c, d) Raw 264.7 and (e, f) 3T3-L1 undifferentiated cells. Data were collected from three independent experiments
Fig 3: Expression and location of elastolytic enzymes in the aorta and PVAT. Expression of genes for cathepsins and MMPs in the a aorta (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively) and b PVAT (n = 3, 5, and 6 for control, ob/ob, ob/ob + Piog groups respectively). mRNA amount is expressed relative to the average expression in control mice. *P < 0.05, **P < 0.01, and ***P < 0.001. ND, not detectable. c Immunofluorescence staining for CTSS (green), MMP-12 (red) and F4/80 (blue) in the thoracic aorta and PVAT. The DAPI nuclear counterstain appears light blue. Magnification of the square in (c) is shown in (d). The while arrow indicates the location of elastic fiber break with increased signals of MMP-12 and F4/80. e Plasma levels of CTSS, MMP-12, and MCP-1 by ELISA (n = 7, 5, and 6 for control, ob/ob, and ob/ob + Piog groups respectively). All data are resulted from 3-month-old male mice. Original magnification × 400 for (c) and × 1200 for (d)
Fig 4: Effect of PPAR? activation on expression of Ctss and Mmp12. a Raw 264.7 cells, b peritoneal macrophages, c 3T3-L1 undifferentiated cells, d 3T3-L1 differentiated cells, and e A7r5 cells were treated with various concentrations of pioglitazone for 20 h. Expression of Ctss and Mmp12 relative to the average expression of non-treatment control group. Data were collected from two independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001
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