Propyl gallate

Human fibrosarcoma cell line HT1080 was grown in Dulbecco’s Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin–streptomycin solution, at 37 °C under a 5% CO₂ atmosphere. All reagents were obtained from commercial sources: cisplatin (CDDP), N-acetylcysteine (NAC), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (WAKO, Osaka, Japan), propyl gallate (Sigma, St. Louis, MO, USA), and Z-VAD-fmk (peptide institute, Osaka, Japan). The antibodies used were against FLAG (Sigma), LKB1, total AMPKα, caspase-3 (Santa Cruz, Dallas, USA), phospho-p38 (threonine 180 and tyrosine 182), total p38, phospho-JNK (threonine 183 and tyrosine 185), total JNK, p53 (Cell signaling technology, Danvers, USA), and β-actin (Wako).

All chemicals were of the highest purity available and used as received. Gallic acid monohydrate (98.0% pure) and gallates (propyl gallate, PG, butyl gallate, BG, octyl gallate, OG, and lauryl gallate, LG, all 99.0% pure) were purchased from Sigma–Aldrich. 2–Hydroxypropyl–β–cyclodextrin (HP–β–CD) and methyl–β–cyclodextrin (M–β–CD) were purchased form Cyclolab LTD and acetonitrile was received from (ACN, HLPC grade) Merck. The Folin-Ciocalteu’s reagent was obtained by VWR Chemical. Sodium carbonate and 2,2–diphenyl–1–pycrilhydrazyl (DPPH^●) were purchased from Sigma–Aldrich. Milli-Q grade water (conductivity < 0.1 mS cm⁻¹) was employed in the preparation of aqueous solutions. Citric acid and sodium citrate (both from Acros Organics, 99% pure) 0.04 M, pH 3.65, was used as buffer solution.

All chemicals were of the highest purity available and used as received. The AC was extra pure charcoal activated powder from Merck, Catalog No K49794484816 (BET specific Surface area (m²/g) 800; particle size (nm) 50–150, >80%; apparent density 150–440 kg/m³). Gallic acid monohydrate and propyl gallate were both 98.0% pure, purchased from Sigma-Aldrich and Fluka, respectively. Folin-Ciocalteu’s reagent was obtained by VWR Chemical. Sodium carbonate and 2,2-diphenyl-1-pycrilhydrazyl (DPPH^•) were purchased from Sigma-Aldrich. Milli-Q grade water was employed in the preparation of aqueous solutions. All materials and culture media used for the establishment of in vitro culture were previously autoclaved at 98 kPa and T = (121 ± 1) °C for 20 minutes.

Cryosections (10 μm) from adult hearts were fixed with 4% w/v paraformaldehyde (pH 7.4) for 5 min at room temperature and were then permeabilized in 0.1% v/v Triton X-100 (Sigma) for 4 min. Following this, sections were covered in blocking buffer solution (10% v/v goat serum [Gibo] and 1% w/v bovine serum albumin [BSA; Thermo Fisher Scientific] in PBS) for 1 h at room temperature. After 1 h, blocking buffer solution was removed and replaced with blocking buffer containing primary antibodies for Connexin40 (Cx40) (1:50, Alpha Diagnostics, Cat# IGG1 CX40A), Sarcomeric myosin (MF20) (1:50, Developmental Studies Hybridoma Bank, Cat# MF-20). Slides were then washed with PBS three times for 3 min each and were then incubated with secondary goat anti-mouse antibody conjugated to Alexa Fluor 488 (1:200, Invitrogen) and goat anti-rabbit antibody conjugated to Alexa Fluor 555 (1:200, Invitrogen) in blocking buffer for 1 hour. Nuclei were counterstained by immersion of a solution containing 1 μg/ml Hoechst 33258 (Sigma) in PBS. Slides were mounted with 0.1% propyl gallate (Sigma) solution (0.1% w/v propyl gallate and 50% v/v glycerol [Thermo Fisher Scientific] in PBS) and were observed under a laser scanning confocal microscope (Zeiss LSM 710).

Dihydroconiferyl alcohol (DCA, 98%), octanoic acid (≥98%), decanoic acid (≥98%), lauric acid (for synthesis), tert-butanol (≥99%), propyl gallate (PG, ≥98%), ethyl acetate (≥99.5%), acetonitrile (≥99.9%), 2-Chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP), N-Hydroxy-5-norbornene-2,3-dicarboxylic acid imide (NHND, 97%), 5% Pd/C and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were purchased from Sigma-Aldrich. (Hoeilaart, Belgium). Immobilized lipase B from C. antarctica (Novozym^® 435) was a gift from Novozymes (Bagsvaerd, Denmark). Molecular sieves (3 Å, 1.6–2.5 mm, technical, water absorption capacity 0.2 g/g) were acquired at VWR. Hexanoic acid (98%) and silica gel 60 (42–60 µm, 230–400 mesh) were purchased from Merck (Hoeilaart, Belgium). Petroleum ether (b.p = 40–60 °C), butylated hydroxytoluene (BHT, 99.8%) and butylated hydroxyanisole (BHA, 98%) were purchased from Acros Organics (Geel, Belgium).
Technical organosolv lignins were supplied by CIMV (botanical origin: wheat straw) and Fraunhofer (botanical origin: beech wood). These lignins were depolymerized as described in Section 2.6.

The solvents used in this study were obtained from VWR International s.r.l. (Milan, Italy). Gallic acid, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, ellagic acid, quercetin, catechin, rutin, ascorbic acid, propyl gallate, butylated hydroxytoluene (BHT), β-carotene, linoleic acid, pancreatic lipase, Tween 20, sodium potassium tartrate, sodium chloride, sodium carbonate, Folin-Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ), o-dianisidine (DIAN) color reagent, peroxidase-glucose oxidase (PGO), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, (ABTS) solution, sodium acetate, β-carotene, linoleic acid, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Dulbecco’s Modified Eagle Medium (DMEM), dimethyl sulfoxide (DMSO), and Fetal Bovine Serum (FBS) were purchased from Sigma-Aldrich s.r.l. (Milan, Italy). l-Glutamine and penicillin/streptomycin were purchased from Gibco, Life Technologies (Waltham, MA, USA).

PKH26-dyed EVs were incubated with either serum from F. hepatica-infected rats (0, 7, 21 and 70 days post-infection) or anti-EV antibodies at 1:100 dilution at 4°C overnight. Unbound antibodies were removed by centrifugation and the EV pellets were re-suspended in PBS. RAW264.7 cells were allowed to attach to μ-Slide 8-well chambered coverslips (Ibidi) overnight and the following day they were transferred to DMEM supplemented with exosome-depleted FBS (Gibco) and 1% L-glutamine. Cells were incubated with 5 μg per well of PKH26-dyed EVs (control) or antibody-coated EVs for 3 hours at 37°C with 5% CO₂. In some experiments, cells were incubated with EVs in the presence of 2μg/ml cytochalasin D (ThermoFisher Scientific). Cells were washed three times in PBS and then fixed with 4% PFA, washed in PBS, counterstained with DAPI and mounted in glycerol containing 10% (v/v) PBS and 0.1 M propyl gallate (Sigma-Aldrich). Slides were examined using a Leica SP5 confocal microscope (Leica Microsystems) with LAS AF software (Leica). Fluorescence was quantified using ImageJ. At least 8 fields were analysed per experiment and a 1-way ANOVA was performed.