Mc lr

Cells were washed twice in ice-cold PBS without calcium or magnesium, fixed in 4% paraformaldehyde (Thermo Fisher Scientific), and permeabilized with 0.1% Triton X-100 (Thermo Fisher Scientific). Cells were then incubated in 3% BSA (Sigma-Aldrich) to block cross-reactivity, and probed with specific primary antibodies for overnight at 4°C. The following primary antibodies were used at the indicated dilutions: MC-LR (Alexis Biochemicals, 1:200), vimentin (CST, 1:200), E-cadherin (CST, 1:200), fibronectin (Abcam, 1:200), and αSMA (Abcam, 1:200). The next day, cells were washed and stained with Alexa Fluor 488- and Alexa Fluor 549-labeled secondary antibodies (Thermo Fisher Scientific, 1:300) for 1 h at room temperature in the dark. Nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml in PBS, Thermo Fisher Scientific) for 10 min at room temperature. Cells were washed, mounted onto glass slides, and visualized using an Olympus confocal laser scanning microscope imaging system and a Zeiss fluorescent microscope.

MC‐LR was acquired from Alexis Biochemicals.

Microcystin standards (MC-LR, MC-RR and MC-YR) with purities ≥95% were purchased from Alexis Biochemicals (Lausen, Switzerland). The stock solution of three MC congeners was prepared in 1 mL of Methanol solution at a concentration of 100 mg/L for each variant, which was kept at −20 °C for later use. The C18 solid phase extraction (SPE) cartridges (500 mg, 6 mL) used in MCs analysis were obtained from Anpel Company (Shanghai, China). The standard compounds GEO, MIB and 2-isobutyl-3-methoxypyrazine (IBMP, as the internal standard) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) with the concentration of 100 mg/L in Methanol, while CYC and ION with purities ≥97% were purchased from Adamas Reagent Co., Ltd. (Basel, Switzerland). The stock solution of 1 mg/L with four target T&O compounds prepared in Methanol was stored in the dark at 4 °C. Sodium chloride (NaCl, Sigma, St. Louis, MO, USA) was added to the samples to enhance the T&O compounds extraction from water.
Methanol, trifluoracetic acid, acetic acid, acetonitrile and acetone were of HPLC grade from Tedia Company (Fairfield, OH, USA). Water used throughout the work was from a Milli-Q water purification system (Millipore, Billerica, MA, USA). All other reagents used were of analytical grade or better.