Fdx 35

The assay was carried out as previous reported with modification to determine the antiangiogenic activity of HLBT-100 [63 (link), 79 (link)]. Briefly, a 170 g (5–6 weeks old) Sprague-Dawley rat (Harlan, Frederick, Maryland) was euthanized by CO₂ asphyxiation. All rats used were maintained in the vivarium at the Institute of Human Virology at the University of Maryland, School of Medicine in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines. The aorta was dissected using a dissecting microscope and the periaortic fibroadipose tissue was removed and 1–2 mm long aortic rings were sectioned and rinsed extensively in EBM media (endothelial cell basal media). The rings were embedded in 200 µl of matrigel in 24-well culture plates so that the lumen was parallel to the base of the plate. 800 µl of EBM without ECGS was added to each well. The rings were incubated for 24 h in an incubator at 37 °C and 5% CO₂ in humidified air and the media was replaced with fresh 800 µl of EBM with ECGS having varying concentrations of HLBT-100. The rings were further incubated for 4–5 days and evaluated for sprout formation. Capillary sprout formation compared to control were captured with a Nikon FDX-35 camera mounted onto a Nikon Eclipse TE300 microscope.

Both Hepa1-6 and HepG2 hepatoma cells (of mouse and human origin, respectively) were cultured in Dulbecco's MEM supplemented with 10% fetal bovine serum (FBS), 2mM L-Glutamine and 100U penicillin/streptomycin mix as previously described [26 (link),27 (link)]. Cells were passaged every 3-4 days and cultured in 6 wells plates for imaging experiments. For treatment with free fatty acids, cells were exposed 24 hours to a mixture of albumin conjugated oleic acid and linoleic acid (OA/LA, Sigma) to a final concentration of 100 μM. Constructs for macroH2A1.1 and macroH2A1.2 were previously described [16 (link), 44 (link)]. Transient transfections were performed using Lipofectamine reagent (Invitrogen), according to manufacturer's instructions. Oil Red O (ORO) staining of lipid droplets was performed as previously described [26 (link)]. Fluorescent images were taken on a Nikon Eclipse TE200 inverted fluorescent microscope, using a U-III advanced exposure system with Multi-point sensor, and a Nikon FDX-35 camera. Filters used were i) G-2A; Ex510-560; DM 575; BA590 (Red); ii) UV-2A; Ex 330-380; DM 400; BA 420 (Dapi): Dapi-Fitc-Rhodamine (Cherry and merge). Software ImageJ (NIH) was used to quantify ORO-stained lipid droplets in individual Hepa1-6 or HepG2 cells [26 (link)].

In order to reveal the microstructure, a standard metallographic technique was used, consisting of its grinding with SiC paper sheet, polishing to a mirror finish and etching with Kroll reagent (HF + HNO₃ and deionized water, 1:3:96) [9 ]. The microstructure was observed with a Nikon EPIPHOT 300 optical microscope coupled to a Nikon FDX-35 camera (Nikon Instruments Europe B.V., Amsterdam, The Netherlands).

Liver sections were processed and analyzed as previously described^{46 (link)}. Briefly, liver fragments were frozen in n-hexane with liquid nitrogen, and cryostat sections (12 μm) were mounted onto aminopropyltriethoxysilane-coated glass slides. From each block, exhaustive 12-μm serial sections were obtained (with 200 μm between sections) and randomly selected for analysis, and at least 3 sections were analyzed. Glass slides with the sections were incubated with Oil Red O for 5 min at room temperature and then rinsed with water. After rinsing, glass slides were coated with water-soluble mounting medium and coverslips. Images (four different fields of each section) were captured using a bright field Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) equipped with a digital camera (Nikon FDX-35, Nikon, Tokyo, Japan). Analysis was performed using the free software Image J (http://imagej.nih.gov/ij). Colored images were converted to grayscale and densitometry was performed on the entire captured field. The section staining and image acquisition were performed in a single experimental round and analysis was performed assuming a constant background signal for all images. Data are presented as Oil Red O optical density.

The samples were prepared for microstructural observation following a standard metallographic technique: smoothing with 400-, 600-, 1200- and 1500-grit SiC paper sheet, polishing with 0.3 μm alumina to a mirror finish and chemical etch with Kroll’s reagent (HF + HNO₃ and distilled water in 1:3:96 proportions) [34 ]. The microstructure was characterized using a Nikon EPIPHOT 300 optical microscope coupled to a Nikon FDX-35 camera (Nikon Instruments Europe B.V., Amsterdam, The Netherlands).