Dp20 5

The liver sections were fixed with 10% formalin and embedded in paraffin. Subsequently, 5-µm-thick paraffin-embedded sections were stained with hematoxylin and eosin. Liver steatosis and inflammation were evaluated based on a previously described scoring system [32 (link)]. The liver sections were also stained with Sirius Red. For immunohistochemistry, the sections were pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6.0) for 20 min. As the primary antibodies, alpha-smooth muscle actin (α-SMA) (ab5494; 1:200 dilution, Abcam, Cambridge, UK) and F4/80 (ab100790; 1:100 dilution, Abcam) were used with staining performed according to the suppliers’ recommendations. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system (Vector Laboratories, Burlingame, CA, USA). DAB was used as the chromogen. Specimens for histology and immunohistochemistry were observed under an optical microscope (BX53; OLYMPUS, Tokyo, Japan) equipped with a digital microscope camera (DP20-5; OLYMPUS). The National Institutes of Health (NIH) ImageJ software (http://imagej.nih.gov/ij/) was used for quantitative analyses. All quantitative analyses were performed for 5 fields per each section in high-power fields (HPFs) at 400-fold magnification.

HUVECs (passages < 12, 1.5 × 10⁵/0.5 ml) were seeded in a 24-well plate and transfected with either microRNA mimics (10 nM), siRNAs (10 nM) or Tough Decoy (50 nM) on the next day. Forty-eight hours after transfection, tube formation assay was performed. HUVECs were plated (6.5 × 10⁴ cells/0.5 ml) into Matrigel-coated wells in a 24-well plate to induce tube networking. BD matrigel matrix was purchased from BD Biosciences (Bedford, MA). Twenty-four hours later, the wells were examined with an Olympus CKX41 microscope and images were captured using an Olympus DP20–5 digital camera. The capillary tubes in those images were counted. For analysis of TuD transfection, images were taken eight hours after transfection instead of 24 hours after. For inhibitor assays, Ki8751 (Calbiochem, San Diego, CA) or N–[(3,5-difluorophenyl) acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl ester-glycine (DAPT; Peptide Institute) was added during tube formation on Matrigel at the indicated concentrations. To recover cells from Matrigel-coated wells, the cells were washed with PBS and incubated with 1 ml of ice-cold BD Cell Recovery Solution (BD Biosciences) with agitation on ice until the gel was resolved. Collected cells were washed with PBS twice and subjected to further analysis.