For the treatment study, αVEGFR2 treatment was given 2 weeks after starting the MCD diet, when mice already developed steatosis, inflammation, and ballooning. Mice were sacrificed under isoflurane anesthesia (Forene, Hoofddorp, The Netherlands) while blood was obtained from the carotid artery. The liver and spleen were rapidly excised and weighed.

The left liver lobe was fixed in 4% phosphate buffered formaldehyde (Klinipath, Olen, Belgium). The right liver lobe was collected in RNAlater (Qiagen, Venlo, The Netherlands) and snap-frozen in liquid nitrogen. The left liver lobe was embedded in paraffin, histologically processed, and sections were cut and stained with hematoxylin and eosin staining (H&E) and Sirius Red. All stainings were performed using standard histology protocols and evaluated by an Talazoparib concentration experienced pathologist. Z-IETD-FMK price The degree of steatosis, lobular inflammation, and ballooning were defined as stated previously.19 Degree of steatosis, defined as the percentage of hepatocytes containing fat droplets, was scored using the following scale: 0 (<5%), 1 (5%-33%), 2 (>33%-66%), 3 (>66%). Foci of lobular inflammation were defined as two or more inflammatory foci (averaged from 3-4 200×

fields) and scored as: 0 (no foci), 1 (<2 foci), 2 (2-4 foci), 3 (>4 foci). Ballooning was scored according to number of ballooned hepatocytes: 0 (none), 1 (few), 2 (many). The degree of fibrosis was evaluated separately and scored as: 0 (none), 1 (zone 3 perisinusoidal or portal fibrosis), 2 (zone 3 perisinusoidal and periportal fibrosis without bridging), 3 (bridging fibrosis), 4 (cirrhosis). Primary hepatocytes were prepared by in situ perfusion and collagenase Venetoclax digestion (Liberase Blendzymes, Roche) of livers of adult female C57BL/6 mice as described.20 Cells were plated at a density of 2.5 × 104 cells per well on collagen I-coated 96-well plates (Greiner Bio-One, Frickenhausen, Germany) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10% FBS, ITS (insulin

5 μg/mL, transferrin 5 μg/mL, selenium 5 ng/mL) and 1% streptomycin and penicillin in 5% CO2 at 37°C. Two hours after plating, medium was replaced by the same culture medium but without ITS. The cells were used for experiments after an overnight incubation.21 Oleic and palmitic acids stock solutions (100 mM) were prepared in 0.1 M NaOH at 70°C as previously described.20 A 5 mM free fatty acid (FFA) / 5% bovine serum albumin (BSA) working solution was prepared by complexing an appropriate volume of stock solution to 5% BSA (FFA-free low endotoxin; Sigma-Aldrich, Bornem, Belgium) in a 60°C water bath. After filtration and cooling, a mixture of oleic and palmitic acids was prepared at a molar ratio of 2:1. After a 3-hour serum deprivation, hepatocytes were treated 24 hours with 100 μg/mL IgG, 100 μg/mL αVEGFR2, 150 ng/mL VEGF, or 100 μg/mL αVEGFR2 and 150 ng/mL VEGF diluted in DMEM/F12.