Fumonisin b1

LC–MS grade methanol, formic acid and QuEChERS [41 (link)] kit were purchased from Sigma Aldrich. Deionised water, water purified through Milli-Q system (Millipore) was used to prepare the standard solutions and for LC/MS analysis. The following mycotoxin standards were used: aflatoxins mix of AFG1, AFB1, AFG2, AFB2 (10 mg / L), ochratoxin A (OTA, 10 mg / L), ochratoxin B (OTB, 10 mg / L) fumonisin B1 (FB1, 50 mg / L), fumonisin B2 (FB2, 50 mg / L) were procured from Sigma Aldrich.

Cell viability was determined by quantitation of ATP, an indicator of live cells, using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) kit, as described previously [32 (link), 58 (link)]. Briefly, cells (4000 cells/well) were grown in 96-well plates with 10% FBS supplemented RPMI-1640 medium. Cells were treated with test agents in 5% FBS medium for 72 hours. Cell viability was determined by the measurement of ATP in a Synergy HT microplate reader (BioTek, Winnooski, VT, USA), following incubation with CellTiter-Glo reagent. For combination treatment, cells (3×10⁶ /100-mm dish; 4000 cells/well in 96-well plates) were grown in 10% FBS RPMI-1640 medium overnight and then cultured in 5% FBS medium containing d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol HCl (PDMP; 5 μM) or fumonisin B1 (FB1, 25 μM) and doxorubicin (Dox; 5∼2.0 μM) for 48 hr. PDMP was purchased from Matreya (State College, PA) and fumonisin B1 (FB1) from Sigma-Aldrich (St. Louis, MO).

tdTomato and Lifeact-GFP were purchased from ibidi, Inc. Ser3 peptides with the sequence of MAS(p)GVAVSDGVIKVFN were synthesized by GenScript (GenScript). GW4869, desipramine, fumonisin B1, C2-ceramide, and Pyrazolopyrimidine 2 (PP2) were purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml. After 14–18 days in vitro (DIV), neurons were treated at 37 °C with 50 ng/ml IL-1β, with control neurons receiving equal volumes of vehicle. cLTP was induced as described previously [24 (link), 25 (link)]. Briefly, hippocampal neurons were maintained in normal ACSF (5 mM HEPES [pH 7.3], 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 33 mM glucose). Osmolarity was adjusted to 290 mosmol/l. Chemical LTP was induced by changing the medium to Mg²⁺-free ACSF (5 mM HEPES [pH 7.3], 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 33 mM glucose, 0.2 mM glycine, 0.02 mM bicuculline, and 0.003 mM strychnine) for 10 min. After that, the incubation solution was altered back to control solution without glycine for 20 min before surface GluA1 labeling and for 30 min before fixation for immunohistochemistry to detect changes in F-actin, respectively. Neurons were treated with vehicle or IL-1β before (1 h), during (10 min), and after cLTP stimulation at indicated time points.

As protease inhibitors, EDTA-2Na (purity: >99.5%) and PMSF (purity: >98.5%), were purchased from Dojindo Laboratories (Kumamoto, Japan) and Sigma-Aldrich Japan (Tokyo, Japan), respectively. Acetonitrile (ACN, LC/MS grade, purity: 99.8%), methanol (MeOH, LC/MS grade, purity: 99.7%), ethanol (EtOH, special grade, purity: 99.5%), formic acid (FA, LC/MS grade, purity: 99.5%), acetic acid (AcOH, LC/MS grade, purity: 99.5%), trifluoroacetic acid (TFA, special grade, purity: 98.0%), ammonium carbonate (special grade), and 28% ammonia solution (NH₄OH, special grade) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Water used for the preparation of all the solutions was purified using a Milli-Q apparatus (Millipore, Billerica, MA, USA); LC/MS analysis used ultrapure water from Wako. The mycotoxins (ochratoxin A, fumonisin B1, zearalenone, deoxynivalenol, patulin) were purchased from Sigma (St. Louis, MO, USA). Drugs with amide, ester, and sulfonamide bonds and amino acid-containing compounds were obtained from the following companies: Aldrich and Sigma Japan (Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), Tokyo Chemical Industry (Tokyo, Japan), and Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, ≥99%), sulfo-N-hydroxysulfosuccinimide (sulfo-NHS, ≥98.5%), and SWCNTs were purchased from Sigma Aldrich. Ochratoxin A (OTA), Deoxynivalenol (DON), Fumonisin B1 (FB1), Patulin, Warfarin, Aflatoxin B1 and G1 from Aspergillus flavus ≥ 98.00% (HPLC), and Aflatoxins B2 and G2 ≥ 98.00% (TLC) standards were purchased from Sigma-Aldrich. Ochratoxin B (OTB) was purchased from Santa Cruz Biotechnology Canada. Carboxyl QDs 525 (Green CdSe/ZnS) and Carboxyl QD 655 (Red CdSeTe) were purchased from Life Technologies (Thermo Fisher Scientific), Burlington, ON Canada. All buffers were prepared with Millipore Milli-Q deionized water at 18 MΩ. All molecular biology grade electrophoresis chemicals were purchased from BioShop Canada (Burlington, Ontario). Reference samples (wheat, barley, corn, oats and malted barley) were provided from Trilogy Analytical laboratory. 5′NH₂ - modified A08min and 1.12.2 aptamers were purchased from Integrated DNA Technologies (IDT).

Reference substances of aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), aflatoxin G₁ (AFG₁), aflatoxin G₂ (AFG₂), deoxynivalenol (DON), fumonisin B₁ (FB₁), fumonisin B₂ (FB₂), T-2 toxin (T-2), HT-2 toxin (HT-2), ochratoxin A (OTA), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), and β-zearalanol (β-ZAL) were purchased from Sigma Aldrich Chemicals (St. Louis, MO). Standard stock solutions (100 µg/mL) were diluted to mixed working solutions with 35% methanol and sample matrix solutions. Concentrations of the mixed standard working solutions are shown in Table S3.
Methanol (LC/MS grade) and acetonitrile (LC grade) were purchased from Merck KGaA (Darmstadt, Germany). Formic acid, acetic acid, and ammonium acetate, both of LC/MS grade, were supplied by Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). QuEChERS purifier was obtained from Meizheng Bio-Tech Co., Ltd. (Wuxi, Jiangsu, China), and it consisted of octadecyl silica (C18), silica, graphitized carbon black (GCB), magnesium sulfate (MgSO₄), and primary secondary amine (PSA).

Before treatments, hDPSCs were cultured up to 28 days from the pulp separation. For cell signaling analysis, hDPSCs were stimulated with recPrP^C (0.5 μg/mL) (Jena Bioscience, Jena, Germany) several times (5, 10, 20, 40 min) at 37 °C in 5% CO₂. To understand the implication of lipid rafts in the signaling of recPrP^C, hDPSCs were pretreated with 30 μM Fumonisin B1 (Sigma-Aldrich, Milan, Italy), a compound that blocks the synthesis of sphingolipids, for 24 h at 37 °C or, alternatively, with 5 mM methyl-β-cyclodextrin (MβCD) (Sigma-Aldrich, Milan, Italy), a compound which is known to induce cholesterol efflux from the membrane, for 30 min at 37 °C. Moreover, to assess the role of endogenous PrP^C in recPrP^C signal pathways and neuronal differentiation induction, hDPSCs were pretreated with siRNA PrP for 72 h as described extensively below. To evaluate the role of recPrP^C in neuronal differentiation process, hDPSCs were stimulated with recPrP^C (0.5 μg/mL) for a long exposure time (14 days) changing the media every 4 days.