Arium pro

Niacinamide (NIA), k-C RG, polyvinylpyrrolidone (PVP), jojoba oil, tween 80, oleic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) powder, k-carrageenan, chloroform, jojoba oil, trypsin, and phosphate-buffered saline (PBS) were supplied by Sigma-Aldrich (Alabaster, AL, USA). l-α-phosphatidylcholine (EPC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Ethanol absolute (≥99.8%) and methanol (≥99.8% HPLC grade) were obtained from Fisher Chemical (Thermo Fisher Scientific, Loughborough, UK). Double-deionized water was provided by an ultra-pure water system (Arium Pro, Sartorius AG, Gottingen, Germany). The porcine ears were acquired in a local slaughterhouse (Porto, Portugal). Reagents were weighted in a digital analytical balance Kern ACJ/ACS 80-4 (Kern & Sohn; Balingen, Germany). The pH measurements were achieved using a Crison pH meter GLP 22 with a Crison 52-02 tip (Crison; Barcelona, Spain).

Total HM carbohydrates were analyzed using GPC. Glycans were separated by the GPC stationary phase and eluted according to size and charge. Neutral mono-, di-, and oligosaccharides, and acidic oligosaccharides with different degrees of polymerization (DP), could be detected. HM carbohydrate solution was prepared by dissolving 0.2 g/mL of total HM carbohydrates in ultrapure water (Sartorius Arium Pro) containing 2% (v/v) 2-propanol at 37 °C. Five milliliters of 0.2 µM filter-sterilized HM carbohydrate solution were injected for each GPC run. The sample loop was cleaned by ultrapure water prior to analysis. Two connected Kronlab ECO50 columns (5 × 110 cm) packed with Toyopearl HW 40 (TOSOH BIOSCIENCE) were used. Milli-Q water was maintained at 50 °C using a heating bath (Lauda, RE 206) for column equilibration. Milli-Q water containing 2% (v/v) 2-propanol was used as the eluent. The flow rate of the eluent was set at 1.65 mL/min. Eluting glycans were monitored by refractive index detection (Shodex, RI-101). The resulting chromatograms were analyzed by using Chromeleon^® software (Thermo Scientific).

Sodium alginate was purchased from ACROS Organics^TM (Thermo Fisher Scientific, Massachusetts, MA, USA). Calcein, k-CRG, RV tartrate, and Dulbecco’s phosphate-buffered saline (PBS) (10×) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Exelon^® patches were acquired from Novartis. Acetonitrile was acquired from Fisher Scientific (Hampton, NY, USA). Silicone moulds were obtained from Micropoint Technologies (Pioneer junction, Singapore). Fresh porcine ears obtained from different adult animals were purchased in a local slaughter (Porto, Portugal). Double-deionised water was provided by an ultra-pure water system (Arium Pro, Sartorius AG, Gottingen, Germany). The reagents were weighted in a digital analytical balance Kern ACJ/ACS 80-4 (Kern & Sohn; Balingen, Germany). pH measurements were achieved using a JENWAY 550 pH meter (Staffordshire, UK). Texture analysis was performed using a TA.XT2 Texture Analyser (Stable Micro Systems Ltd., Haslemere, UK). SEM and confocal fluorescence microscopy studies were performed at the Laboratory for Scanning Electron Microscopy and X-Ray Microanalysis and High-Resolution Microscopy Unit, respectively, both at CEMUP (Materials Centre of the University of Porto, “Centro de Estudos de Materiais da Universidade do Porto”). Histological samples have been processed in the histological service of “ICBAS, Universidade do Porto”.

Chestnuts (C) and peanuts (P) were purchased from a local market and the shells were separated manually. Ultrapure water was produced with an Arium© Pro instrument from Sartorius (Goettingen, Germany). Ammonia aqueous solution (NH₄OH, ACS reagent, 28–30% solution) was purchased from Merck. 9,10-Diphenylanthracene (>99%) was purchased from Ega-chemie.

The following small molecules were evaluated for induction or reduction of pathway activity. Wnt: CHIR99021(#SML1046) [36 (link)], LY2090314 (#SML1438) [36 (link)], niclosamide (N3510) [37 (link)], LGK-974 (#S7143) [36 (link)]; Hh: SAG dihydrochloride (SAG, #SML1314) [38 (link)], sonidegib (#S2151) [39 (link)], vismodegib (#S1082) [40 (link)]; Notch: suberohydroxamic acid (SBHA, #390585) [41 (link)], DAPT (#S2215) [42 (link)], Dibenzazepine (DBZ, #S2711) [43 (link)], valproic acid sodium salt (VPA, #P4543) [44 (link)]. All reagents were purchased from Selleckchem (Munich, Germany) or Sigma Aldrich. VPA and SAG were dissolved in ultrapure water (Arium pro, Sartorius AG, Göttingen, Germany), and the remaining compounds in DMSO (#D5879, Sigma-Aldrich).
Transfections and evaluations were performed as described above, with the following changes: For induction of the Wnt-pathway in the respective wells, 100 μl conditioned medium (containing wnt3a, see above) was added to the wells four hours after transfection. Compounds were also added four hours after transfection, at a final concentration of 10μM. An equal amount of appropriate solvent (water or DMSO) was added to the solvent control conditions.

L-ergothioneine (ET; purity > 98%), ET sulfonate (ET-SO₃H), L-hercynine, S-methyl ET, and heavy-labelled L-ergothioneine-d9 (ET-d9) and L-hercynine-d9 were provided by Tetrahedron (Paris, France; www.tetrahedron.fr). Stock solutions (1 mM) were prepared in ultrapure water (Arium pro, Sartorius AG, Gottingen, Germany) and stored at −20 °C. HPLC-grade acetonitrile and methanol, and MS graded formic acid were purchased from Fisher (Hampton, USA), GC-grade acetone from Tedia (Fairfield, USA), Triton X-100 from USB corporation (Cleveland, USA), and phosphate buffered saline (PBS)from Lonza (Basel, Switzerland). All other chemicals were obtained from Sigma - Aldrich (St Louis, USA) unless otherwise stated.

Stock standard solutions containing the seven metabolites investigated (i.e., monoethyl phthalate (MEP); mono-benzyl phthalate (MBzP); mono-butyl phthalate (MBP); mono-cyclohexyl phthalate (MCHP); mono-2-ethylhexyl phthalate (MEHP); mono(3,5,5-trimethylhexyl) phthalate (MTMHP); and mono-n-octyl phthalate (MnOP) (100 mg/L in cyclohexane)) were supplied by Neochema GmbH (Germany). Working solutions for calibrations were prepared by serial dilution of the stock standard mixture with acetonitrile (Sigma-Aldrich, Darmstadt, Germany) Beta-glucuronidase from Escherichia coli (200 units/mL) was purchased from La Roche AG group. BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) with 1% of TMCS (trimethylchlorosilane) was purchased from Sigma-Aldrich. Ammonium acetate (98%), calcium chloride (CaCl₂) (≥99%), creatinine hydrochloride (≥97%), ammonium phosphate dibasic (≥99%), magnesium chloride hexahydrate (≥99%), potassium chloride (≥99%), sodium sulfate (≥99%), urea (≥98%), acetone (99.9%), formic acid (≥95%), and methanol (99.9%) were purchased from Sigma-Aldrich. Nanopure water was provided by an ultrapure water system (Arium Pro, Sartorius, Göttingen, Germany).