Multi well plates

Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM) and penicillin-streptomycin solution were purchased from Gibco Laboratory (Carlsbad, CA, USA). Trizol reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratorise (Kumamoto, Japan). catechin (> 97%), 3-isobutyl-1-methylxanthine (IBMX), insulin, dexamethasone (DEX), TNF-α and Oil Red O were ordered from Sigma-Aldrich Company (St. Louis, MO, USA). HyPure^TM Molecular Biology Grade Water, phosphate buffer solution (PBS) were purchased from Hyclone (Waltham, MA, USA). The reagents for electrophoretic mobility shift assays were from SunShine Biotechnology (Hangzhou, China). RevertAid First Strand cDNA Synthesis Kit was from Thermo Scientific (Waltham, MA, USA). FastStart Universal SYBR Green Master (Rox) was from Roche (Basel, Switzerland). Antibodies for NF-κB-p65, p-NF-κB-p65 (Ser276) were from Abcam Inc. (Cambridge, MA); SIRT1, p-SIRT1 (Ser27), FOXO3a, p-FOXO3a (Ser253) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from ABclonal Biotechnology (Woburn, MA, USA); AMPK and p-AMPK(Thr172) were from Cell Signaling Technology (Danvers, MA, USA). Cell culture dishes and multi-well plates were supplied by Nunc (Denmark).

VGF assays and their characterization are summarized in Table 1. Competitive ELISA was performed as previously reported [25 (link), 27 (link)]. Briefly, multiwell plates (Nunc, Milan, Italy) were coated with the relevant synthetic peptide (3 h, at room temperature), hence treated with PBS containing 9% normal donkey serum, 20 nM aprotinin, and 1 mg/mL ethylenediaminetetraacetic acid (EDTA) for 2 hours. Primary incubations were carried out in duplicate, including serial dilutions of standard (0.005–500 pmol/ml) or samples (4 h at room temperature). Biotinylated secondary antibodies (Jackson, West Grove, PA), streptavidin-peroxidase conjugate (Biospa, Milan, Italy), and a tetrametylbenzidine substrate (TMB X-traKem-En-Tec, Taastrup, Denmank) were used to reveal bound antibodies. The reaction was stopped with HCl (1 mol/L) and the optical density was measured at 450 nm using a multilabel plate reader (Chameleon: Hidex, Turku, Finland). Recovery of synthetic peptide/s added to plasma, or to tissue samples at extraction was >85% for all assays used. Absolute numerical values of tissue concentrations measured per each peptide studied ought to be considered (or compared across different peptides) with caution, pending precise characterization of the heterogeneous molecular species present.

The cells (5 × 10⁵) were seeded in multi-well plates (Nunc). The following day, the cells were treated with DOX with or without the selective inhibitor ABT-199 of Bcl-2 protein (Cayman Chemical, Ann Arbor, MI, USA) or TNFα (Thermo Fisher Scientific) for 48 h. The percentages of apoptotic cells were determined using the Dead Cell Apoptosis Kit (Thermo Fisher Scientific). Fluorescence was measured on a BD FACS Lyric (Becton-Dickinson, Franklin Lakes, NJ, USA) and data were processed using the BD FACSuite™ Software v1.5.0.925 (Becton-Dickinson). The cells in early apoptosis were stained with Annexin V APC, whereas cells in late apoptosis were stained with Annexin V APC and SYTOX Green.

HepG2 cells, a human hepatocyte-derived cell line, were obtained from ATCC (Rockville, MD, USA). Cells were cultured at 37 °C in a humidified atmosphere with 5% CO₂ in Minimum Essential Medium (MEM) containing 1 g/L glucose, 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin/streptomycin. Cells were cultured in 75 cm² flasks (Thermo Fisher Scientific, Waltham, MA, USA) and plated in either 6 or 12 multi-well plates (Thermo Fisher Scientific, Waltham, MA, USA), at a density of 1 × 10⁶ or a 5 × 10⁵ cells/well, respectively. When reaching 70–80% confluency, cells were subcultured using Trypsin-EDTA solution. To induce lipid deposition, cells were treated for 24 h with 0.5 mM palmitate (Sigma, St. Louis, MO, USA) coupled to fatty acid-free bovine serum albumin (BSA) (Calbiochem, San Diego, CA, USA) in serum-free MEM, as an established in-vitro model of steatosis. Uncoupled BSA (1%) was used in control conditions. ER stress was induced by incubating the cells with thapsigargin (Tg) for 24 h. Dimethylsulfoxide (DMSO) alone was used as a control treatment. Cell viability was assessed by the Alamar Blue assay to determine the toxicity of the compounds employed. No significant differences in cell viability were observed in HepG2 cells exposed to any of these compounds (data not shown).

After three washes, washed MV pellets were re-suspended in 100 µl HEPES (10 mM, NaCl 140 mM, pH 7.40). Multi-well plates (Thermo Fisher Scientific, Illkirch, France) were coated with 150 µl assay buffer made of 50 mM phosphate, 80 mM NaCl, 2 g/L bovine serum albumin (Eurobio, Courtaboeuf, France) and 0.01% Tween. Fibrinolytic MV activity was assessed on 1/1 MV–plasminogen/CBS0065 samples (plasminogen: South Bend, USA; CBS0065: Stago, Asnières-sur-Seine, France). Microvesicle-free plasma (pellet supernatant) was used a negative control, and urokinase (Euromedica, Diegem, Belgium) was used with a range from 0.005 U to 0.5 U. Absorbance variations were measured for 18 h at 405 and 490 nm at 37 °C with a thermostated spectrophotometer (Versamax). Results are expressed in urokinase concentration.