Mc lr

The material used in this study was Microcystin-leucine arginine (MC-LR) (ALX-350-012-M001; Enzo Life Sciences, NY, USA). To prepare the concentrate solution, 1 mg of MC-LR was suspended in 1000 μL of dimethyl sulfoxide (DMSO), yielding a concentration of 1 mM. To obtain 1, 10, 100, and 1000 nM of MC-LR for each experiment, the concentrate solution was diluted with the completed DMEM medium. These concentrations of MC-LR for treatment groups were selected based on microcystin-polluted surface waters, typically below 100 μg/L (equivalent to approximately 100.5 nM) [29 (link)], as well as many previous studies [23 (link),[30] (link), [31] (link), [32] (link)] that have utilized this concentration range.
The β-catenin inhibitor, MSAB (HY-120697; MedChem Express, NJ, USA) was first diluted with DMSO to prepare the stock solution. For the working solution, the inhibitor was further diluted with completed DMEM medium to achieve a 10 μM of MSAB. The inhibitor was treated to the cultured cell 24 h before MC-LR treatment.

Tissue lysates (20 µg/well) were prepared in Laemmli sample buffer with 2.5% BME and heated at 37 °C for 30 min. Protein was transferred from the gel to polyvinylidene fluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System at 25 V and 1.0 A for 30 min. Following transfer, the membranes were imaged under UV to capture Stain-Free image used for protein normalization. The blots were then blocked with 5% non-fat dry milk in Tris-base buffered saline-Tween 20 (TBS-T) for 1 h at room temperature, then incubated with primary antibody overnight at 4 °C. Membranes were probed for OATP1B2 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat. 376904) and MCLR (1:2000 dilution; Enzo, Farmingdale, NY, USA, Cat. 89154-022). The blots were incubated with secondary antibody in 5% non-fat dry milk in TBS-T for 1 h at room temperature. Densitometry was performed using Image Lab (Bio-Rad, Standard Edition, Version 6.0.0 build 25). Proteins of interest were normalized to total protein as captured by Stain-Free image. Total protein normalization is a commonly accepted technique for protein densitometry analysis instead of the single-protein loading control [101 (link)].

Inorganic chemicals and organic solvents were reagent grade or better. Standard of MC-LR for the multihapten-ELISA were from Enzo Life Sciences Inc. (Farmingdale, NY, USA), and this was in-house calibrated against a CRM-MCLR from NRC (Halifax, NS, Canada). The standard for the Adda-ELISA was as provided with the kit (Abraxis LLC, Warminister, PA, USA).

SPF male APC^min/+ C57BL/6 mice (aged 6–7 weeks, 20–22 g) were obtained from GemPharmatech Co., Ltd. (Nanjing, China) and maintained at the Animal Core Facility of Nanjing Medical University. The mice were nourished with a standard rodent diet and maintained in a controlled environment with a 12:12 h light–dark cycle. After adapting to the environment for 2 weeks, 24 mice were randomly and equally assigned to two groups: a treatment group (gavage with 40 µg/kg MC-LR dissolved in 0.9% NaCl solution) and a control group (gavage with the same 0.9% NaCl solution based on mice body weight). MC-LR, with a purity exceeding 95%, was procured from Enzo Life Sciences (Farmingdale, NY, USA). The mice received either the 0.9% NaCl solution or MC-LR by oral gavage for 8 weeks. Body weights were measured daily.

In triplicate, 200 mL of water sample was filtered (CFC, Whatman, Maidstone, UK), filter papers were wrapped individually in aluminium foil and preserved at −80 °C. On analysis, filter papers were subjected to three cycles of freeze-thawing before submersion in 10 mL of 80% aqueous methanol. Samples were left in the dark at 4–6 °C for 24 h, before ~0.5 mL was aliquoted into a LCMS certified vial. Toxin analysis was carried by ultra-high-performance liquid chromatography (UHPLC) (Acquity, Waters, Manchester, UK) coupled to a tandem quadruple mass spectrometer (Xevo TQ, Waters, Manchester, UK). All instrument solvents and chemicals were of LC-MS-grade (Fisher Optima, Thermo Fisher, Manchester, UK). Reference toxins used for the detection method included the microcystin analogues MC-RR, MC-LA, MC-LY, MC-LF, MC-LW, MC-YR, MC-WR, MC-HilR, MC-HtyR, MC-LR & Asp³-MC-LR (Enzo Life Sciences, Exeter, UK) and [Dha⁷]-MC-LR and matrix reference material of blue-green algae (RM-BGA, Lot 201301) containing a range of microcystins (Institute of Biotoxin Metrology, National Research Council Canada). Analysis of microcystins was conducted following the method by Turner et al. [50 (link)].

Gibco (Biomol, Sevilla, Spain) has provided the fetal bovine serum (FBS), minimum essential medium (MEM) and cell culture reagents. HPLC-grade methanol, dichloromethane, trifluoroacetic acid (TFA) and formic acid were obtained from Merck (Darmstadt, Germany). Deionized water (>18 MΩcm-1 resistivity) was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). The MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium salt) Cell Titer 96^® AQueous One Solution Cell Proliferation Assay was purchased in Promega (Biotech Iberica, Madrid, Spain). The neutral red (NR) and the Bradford reagent were obtained from Sigma-Aldrich (Madrid, Spain). BOND ELUT^® Carbon cartridges (500 mg, 6 mL) were supplied by Agilent Technologies (Amstelveen, The Netherlands, Europe). CYN (95%), (+/−) ATX, MC-LR, MC-RR and MC-YR (99%) standards were supplied by Enzo Life Sciences (Lausen, Switzerland).