Zearalenone zea

Potato dextrose (PD) agar and broth, Czapek–Dox (CD) agar and broth, de Man, Rogosa and Sharpe (MRS) agar and broth, malt extract soft (MES) agar, peptone and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from HiMedia, (Mumbai, India). Dichloro-dihydro-fluorescein diacetate (DCFH-DA), α-cyano-4-hydroxy cinnamic acid, trifluoroacetic acid (TFA), methanol, ergosterol and zearalenone (ZEA) were obtained from Sigma–Aldrich (Bangalore, India). All the plasticware used in the study were acquired from Eppendorf (Bangalore, India), and other chemicals and solvents used in the study were fine grade and acquired from Merck Millipore (Bangalore, India).

Zearalenone (ZEA), and 4-phenylbutyrate (4-PBA), were purchased from Sigma–Aldrich (St. Louis, MO, USA); Fetal bovine serum (FBS) and DMEM/F-12 medium were obtained from Gibco (Grand Island, NY, USA); the cell counting kit-8 (CCK8) was purchased from Dojindo Laboratories (Kumamoto, Japan); the LDH release assay, ROS assay kit (S0033) and ATP assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China); and the cell cycle assay caspase 3 activity assay kit and Annexin V/propidium iodide (PI) were purchased from Becton Dickinson Company (BD; Franklin Lakes, NJ, USA). Dorsomorphin dihydrochloride (HY-13418) was purchased from Medchem express (Shanghai, China).

Standard ochratoxin A (OTA), aflatoxin B₁ (AFB₁) and zearalenone (ZEA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Individual stock solutions of mycotoxins were prepared in AcN at 500 µg/mL and formed solutions in MeOH at 100 µg/mL. The solutions were maintained at −20 °C in the dark. Betanin was purchased from Sigma-Aldrich and diluted to prepare solution in EtOH.
To prepare the gastrointestinal solutions, KCl 89.6 g/L, KSCN 20 g/L, NaH₂PO₄ 88.8 g/L, NaSO₄ 57 g/L, NaCl 175.3 g/L, NaHCO₃ 84.7 g/L, urea 20 g/L and Milli-Q water (Milli-Q water purification system (Millipore, Bedford, MA, USA)) were used. First, 145 mg α-amylase in 100 mL of Milli-Q water was prepared. A Pepsin solution with 0.5 mg Pepsin (1 g in 25 mL HCl (0.1 N) (Pepsin from porcine gastric mucosa, powder, ≥250 units/mg solid, P-7000, Sigma-Aldrich, St. Louis, MO, USA) was used. A solution of pancreatin: 1.10 mg (0,1 g Pancreatin (Pancreatin, from Porcine Pancreas, P1750, Sigma-Aldrich, St Louis, MO, USA) and 0.625 g bile salts (Bile extract porcine, B8631, Sigma-Aldrich, St. Louis, MO, USA) in 25 mL of NaHCO₃ (0.1 N) was prepared. The saliva solution was prepared mixing: 10 mL of KCl 89.6 g/L, 10 mL of KSCN 20 g/L, 10 mL of NaH₂PO₄ 88.8 g/L, 10 mL of NaSO4 57 g/L, 1.7 mL of NaCl 175.3 g/L, 20 mL of NaHCO₃ 84.7 g/L, 8 mL of urea (20g/L) and was completed to 500 mL with Milli-Q water.

Methanol and potassium chloride (KCl) were purchased from the Aladdin company (Shanghai, China), potassium ferrocyanide (K₄[Fe(CN)₆]), potassium ferricyanide (K₃[Fe(CN)₆]), disodium hydrogen phosphate dodecahydrate (Na₂HPO₄-12H₂O), disodium hydrogen phosphate dehydrate (NaH₂PO₄-2H₂O), sodium hydroxide (NaOH), and glucose (C₆H₁₂O₆), were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Chloroauric acid (HAuCl₄·4H₂O), graphene oxide nanosheets (GO, XF002-1, 500 nm–5 μm; ~99%, Hummers) were purchased from Nanjing XFNANO Materials Tech. Co., Ltd (Nanjing, China). Tris(2-carboxyethyl) phosphine (TCEP) was obtained from Sigma-Aldric. Ochratoxin A (OTA), ochratoxin B (OTB), deoxynivalenol (DON), and zearalenone (ZEA) were purchased from Sigma-Aldrich (Shanghai, China). Tris(hydroxymethyl)aminomethane hydrochloride (tris-HCl) and ethylenediaminetetraacetic acid disodiumsalt (EDTA) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. OTA binding-aptamer with a sequence (5′-GAT CGG GTGTGG GTG GCG TAA AGG GAG CAT CGG ACA-(CH₂)₆-SH-3′) was purchased from Sangon Biotech. (Shanghai, China). All reagents and chemicals were of the highest analytical grade. Millipore-Q water (18.2 MΩ cm⁻¹) was used to prepare all aqueous solutions.

Zearalenone (ZEA) was purchased from Sigma–Aldrich (St. Louis, MO, USA); resveratrol (RSV) was purchased from Solarbio (Beijing, China, SR8070); DMEM/F-12 medium was obtained from Gibco (Grand Island, NY, USA, 12500-062); fetal bovine serum (FBS) was obtained from Gemini (California, USA, 900-108). RIPA lysate and protease inhibitor complex were purchased from Pulilai (Beijing, China, C1053); GAPDH and SIRT1 antibody were purchased from Cell Signaling Technology (Boston, Massachusetts, USA); GLUT1, LDH, and MCT4 antibodies were purchased from Abcam (Cambridge, MA, USA); LDH release assay was obtained from Beyotime Institute of Biotechnology (Shanghai, China, C0017); the lactic acid assay kit and the pyruvate assay kit were purchased from Nanjing Institute of Biological Research (Nanjing, China, A019-2, A081); and all other reagents and chemicals were analytical grade and were obtained commercially.

To study the functional role of FgStu2 gene, we substituted original FgStu2 promoter with an inducible promoter Pzear (Promoter of the FGSG_04581.3 gene) to generate FgStu2 silent mutant (FgStu-Si) as previously described (Lee et al., 2010 (link)). The schematic of promoter replacement was shown in Figure 4. First, a 1.2 kb geneticin resistance gene (G418) and a 0.8 kb Pzear fragment were amplified from plasmid pNEO and genomic DNA of PH-1 respectively and fused the two fragments to obtain the G418-Pzear fragment by double joint polymerase chain reaction (DJ-PCR) as previously described (Yu et al., 2004 (link); Ren et al., 2014 (link)). Second, a 1.0 kb upstream region of FgStu2 original promoter (Pstu2) and a 1.0 kb FgStu2 coding sequence were amplified from genomic DNA of PH-1. Finally, the three fragments (upstream region of Pstu2, G418-Pzear and 1.0 kb FgStu2 coding sequence) were fused by DJ-PCR to obtain the Pstu2 substitution vector which was used for protoplast transformation. To induce Pzear replacement, 30 μM zearalenone (ZEA, Sigma Aldrich, St. Louis, MO, United States) was added to the medium during the regeneration (Lee et al., 2010 (link)).

Bovine serum albumin (BSA), 3,3′5,5′-tetramethylbenzidine liquid substrate (TMB), and Aflatoxin B1, aflatoxin M1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A (OTA), deoxynivalenol (DON), fumonisin B1 (FB1), and zearalenone (ZEA) standard solutions were purchased from Sigma Aldrich (Merck, Darmstadt, Germany). Methanol (HPLC grade), microplates and all other chemicals were obtained from VWR International (Milan, Italy). Rabbit polyclonal antibodies directed towards Aflatoxin B1 (anti-AFB1) and Aflatoxin B1 conjugated to horse radish peroxidase (AFB1-HRP) were prepared in the laboratory as described in [16 (link)]. Optical density at 450 nm was measured by a Multiskan microplate reader (ThermoScientific, Waltham, MA, USA). Extract were centrifuged in a refrigerated centrifuge (BR, Juan, France).