Naaso2

Endothelial EA.hy926 cells were transferred to 24-well culture plates and cultured until confluent. The cells were treated with arsenite (NaAsO₂, Fujifilm Wako Pure Chemical Co., Ltd.) at 1, 2, 5, 10, or 20 µM and incubated at 37 °C for 24 or 48 h. After treatment, the medium was discarded, and the cells were washed twice with Dulbecco’s phosphate-buffered saline (D-PBS, Fujifilm Wako Pure Chemical Co., Ltd.). The cells were fixed with methanol and stained with Giemsa solution (Merck KGaA, Darmstadt, Germany). The cell layer was observed morphologically using a DMi1 inverted microscope (Leica Microsystem, Wetzlar, Germany). Separately, cell viability was measured using MTT (Dojindo Laboratories, Kumamoto, Japan). Briefly, after treatment with arsenite, the culture medium was changed to fresh 10% FBS-DMEM containing 0.5 mg/ml MTT, and cells were incubated for 4 h at 37 °C. After removing the medium, dimethyl sulfoxide (Fujifilm Wako Pure Chemical Co., Ltd.) was added to MTT formazan. Absorbance at 570 nm was measured by a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

In this study, wild-type (AB strain) and Nrf2-mutant (nfe2l2a^fh318) [15 (link)] zebrafish larvae were used. The Nrf2-mutant line was maintained by polymerase chain reaction based genotyping, as described previously [28 (link)]. Embryos were obtained by natural mating. Capsaicin, carnosic acid, cinnamaldehyde, eugenol, 6-gingerol, isoeugenol, quercetin, H₂O₂ and NaAsO₂ were purchased from FUJIFILM Wako (Osaka, Japan). Diallyl trisulfide and sulforaphane were bought from LKT Laboratories (St. Paul, MN, USA). Curcumin and 6-MSITC were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Abcam (Cambridge, UK), respectively. H₂O₂ and NaAsO₂ were dissolved in MiliQ water (Merck-Millipore, Billerica, MA), sulforaphane in ethanol and other phytochemicals were dissolved in dimethyl sulfoxide for the stock solution, and were diluted to the final concentration with E3+ medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄ and 0.1 μg/mL methylene blue).

NaAsO₂ (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in sterile-filtered water (Sigma-Aldrich) at a concentration of 100 mM. The NaAsO₂ solution (100 mM) was further diluted with culture medium to obtain the indicated concentrations. Primary cultured Fucci-expressing astrocytes were subjected to serum starvation (see Section Fluorescence Microscopy of Fucci-Expressing Astrocytes) to synchronize the cell cycle. After synchronization, the cells were exposed to NaAsO₂ in culture medium at concentrations of 0, 1, 2, or 4 μM. Immediately after starting NaAsO₂ exposure, astrocytes were placed in an incubation chamber (Tokai Hit, Shizuoka, Japan) equipped to the BioZero 8100 fluorescence microscope. In the incubation chamber, the temperature was controlled at 37°C and the CO₂ concentration was maintained at 5%. Time-lapse fluorescence imaging began at 1 h and ended at 73 h after initiation of NaAsO₂ exposure. Fluorescence images of mKO2 and mAG expression, and bright field images were captured every 2 h. The digital image data were obtained from three regions (0.58 mm²/region, 1.74 mm² in total) that were randomly selected in each culture dish compartment. Live imaging of Fucci-expressing astrocytes was performed in six primary cultures derived from different animals.