Additional file 2 of Temporal regulation of notch activation improves arteriovenous fistula maturation

Additional file 2: Table S1. List of antibodies used in this study

IL-6 production is increased in cardiac fibrosis induced by Ang II.

<p>(<b>A</b>) Immunohistochemical analysis of IL-6 production (brown) in ventricles of mice (n = 6 per group) after saline or Ang II infusion. (<b>B</b>) Western blot analysis for IL-6 expression in the hearts of WT mice. (<b>C</b>) Systolic blood pressure (SBP) was measured and echocardiography was performed at day 7 after Ang II infusion. Error bars represent mean±SEM. *<i>P</i><0.05; ***<i>P</i><0.001. Scale bars: 50 µm.</p

IL-6 promotes Ang II-induced cardiac fibrosis and inflammation.

<p>(<b>A</b>) Ang II-induced hypertensive cardiac fibrosis in C57BL/6 wild-type (WT) and IL-6-/- mice (n = 6 per group). (Left) Masson’s trichrome staining (blue) of ventricular sections analyzed for fibrotic areas at day 7 after Ang II infusion (n = 6 mice/group). Scale bars: 500 µm (top), 100 µm (bottom). Fibrotic area is stained blue. (Right) Quantification of fibrotic areas. (<b>B</b>) Western blot analysis and quantification of protein levels of transforming growth factor β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in WT and IL-6-/- hearts (n = 6 per group). Normalization is to GAPDH level. (<b>C</b>) Representative immunohistochemical staining of collagen I, TGF-β1 and α-SMA in WT and IL-6-/- hearts. Scale bars: 50 µm and quantification (right). (<b>D</b>) Immunohistochemical staining and quantification of Mac-2 in WT and IL-6-/- hearts. Scale bars: 50 µm. Data represent the mean±SEM (n = 5 per group). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

Primer sequence for Real time-PCR and RT-PCR.

<p>Primer sequence for Real time-PCR and RT-PCR.</p

Co-culture of macrophages and myofibroblasts induces α-SMA, TGF-β1 and Smad3 expression.

<p>(<b>A</b>) Representative immunohistochemical staining of α-SMA with 3-D co-culture in the present of Ang II. Scale bars: 50 µm. (<b>B</b>) Real-time PCR quantification of α-SMA and TGF-β1 mRNA expression. (<b>C</b>) Immunofluorescence staining of protein level of phosphorylated Smad3 (p-Smad3; green) and DDR2 (red) or Immunofluorescence staining of TGF-β1 (green) and DDR2 (red) in co-cultured WT macrophages and fibroblasts with neutralizing antibody to IL-6 and co-cultured IL-6-/- macrophages and fibroblasts with recombinant IL-6 (rIL-6) treatment. Scale bars: 50 µm. (n = 6 per group). (<b>D</b>) Western blot analysis and quantification of protein level of TGF-β1 and p-Smad3 in 3-D co-culture (n = 4 per group). *<i>P</i><0.05.</p

Generation of inflammatory factors and localization of IL-6 in macrophages or myofibroblasts in Ang II-infused mice.

<p>(<b>A</b>) RT-PCR analysis of mRNA expression of IL-6 in WT mice macrophages, cardiomyocytes and cardiac fibroblasts with Ang II treatment at 0,1,4,12 h respectively. *<i>P</i><0.05 vs. macrophages. (<b>B</b>) IL-6 (green) was detected in WT hearts and in myofibroblasts (red) in Ang II-treated hearts. Nuclei are DAPI stained (blue; right). (<b>C</b>) Macrophages (Mφ) were co-cultured with cardiac fibroblasts (CF), and the level of IL-6 production in the medium was measured by luminex assay (n = 5 per group). *<i>P</i><0.05; ND: no detective; **<i>P</i><0.01 vs. WT macrophages (Mφ(WT)) and cardiac fibroblasts(CF(WT)); **<i>P</i><0.01 vs. Mφ(WT)+cardiac fibroblasts CF(IL-6-/-) and Mφ(IL-6-/-)+CF(IL-6-/-).</p

Cat S deficiency increases the Ang II-induced accumulation of autophagosomes in mouse heart.

<p>Immunostaining for LC3 and F4/80 in (<b>A</b>) mouse heart (Bars, 10 µm) and (<b>B</b>) macrophages (Bars, 5 µm). (<b>C</b>) Western blot analysis of LC3 protein expression in macrophages. (<b>D</b>) RAW 264.7 cells were transfected with Cherry-GFP-LC3, and treated with and without Ang II in the presence or absence of Cat S inhibitor (15 nmol/L). Confocal fluorescence was used to image autophagosomes (red and green foci) and autolysosomes (red-only foci). Bars, 5 µm. (<b>E</b>) Representative electron microphotographs of cytoplasmic vacuolization in macrophages treated with saline or Ang II (100 nM) for 48 hr (n = 3 per group). Bars, 1 µm. (<b>F</b>) Immunostaining for LC3 and mitochondria marker MitoTtracker in macrophages. Bars, 2 µm. Data are mean±SEM (n = 4 per group). *P<0.05 <i>vs.</i> Cat S KO control macrophages.^##<i>P</i><0.01 vs. Ang II-treated WT macrophages. ^§§ P<0.01 <i>vs.</i> WT control macrophages.</p

Cat S deficiency increases Ang II-induced infiltration and expression of proinflammatory cytokines in mouse heart.

<p>(<b>A</b>) Representative hematoxylin and eosin staining of WT and Cat S^−/− hearts. Bars, 50 µm. (<b>B</b>) Immunohistochemical staining and (<b>C</b>) quantification of Mac-2 positivity. Bars, 25 µm. (<b>D</b>) Immunohistochemical staining and (<b>E</b>) quantification of TNF-α and IL-1β expression. Bars, 50 µm. (<b>F</b>) Real-time PCR analysis of mRNA expression of TGF–β1, TNF-α and IL-1β in WT and Cat S KO mouse hearts. Bars, 50 µm. Data are mean±SEM (n = 4 per group). **P<0.01 <i>vs.</i> saline Cat S KO control; ^##P<0.01 <i>vs.</i> Ang II-infused WT mice.</p

Angiotensin II (Ang II) increases cathepsin S (Cat S) expression in mouse heart.

<p>(<b>A</b>) Co-localization of Cat S with F4/80-positive macrophages and (<b>B</b>) α-smooth muscle actin (α-SMA) in WT and Cat S^−/− hearts. Bars, 25 µm. (<b>C)</b> Measurement of blood pressure in WT and Cat S^−/− mice. (<b>D</b>) Left ventricle ejection fraction (EF %) in WT and Cat S^−/− hearts. (<b>E</b>) Immunohistochemical staining of Ang II-induced Cat S expression in mice. Bars, 25 µm. Data are mean±SEM (n = 8 per group). **P<0.01 <i>vs</i>. saline control. ^§§P<0.01 <i>vs</i>. saline control.</p

Cat S deficiency enhances Ang II-induced cardiac fibrosis in mouse heart.

<p>(<b>A</b>) Representative Masson trichrome staining of WT and Cat S^−/− hearts with saline or Ang II infusion and quantitative analysis of fibrotic areas. Immunohistochemical staining and quantification of (<b>B</b>) collagen I, (<b>C</b>) transforming growth factor (TGF)- β1 (<b>D</b>) and (<b>E</b>) α-SMA in WT and Cat S^−/− hearts with saline or Ang II infusion. Bars, 50 µm. Data are mean±SEM (n = 4 per group). **P<0.01 <i>vs.</i> saline Cat S KO control; ^#P<0.05, ^##P<0.01 <i>vs.</i> Ang II-infused WT mice. ^§P<0.05 <i>vs.</i> saline WT control.</p