Isolation and culture of mouse astrocytes:
The experimental protocols followed the ARRIVE guidelines and the National Institutes of Health guidelines for the Care and Use of Animals (NIH Publication No. 8023, revised 1978) and were approved by the Animal Experiment Committee of Kobe Pharmaceutical University (approval number: 2019-17). Astrocytes were prepared from the cerebra of 1-day-old C57BL/6N mice, as described previously (Michinaga et al. 2020). As it was difficult to distinguish between males and females among the 1-day-old mice, a mixed culture of male- and female-derived astrocytes was used in the present study. Isolated cells were seeded at a density of 1 ´ 104 cells/cm2 in 75 cm2 culture flasks and cultured in Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum. To remove oligodendrocyte progenitors and microglia from the protoplasmic cell layer, the culture flasks were shaken at 250 rpm overnight after seeding for 10-14 days. The cell monolayer was then trypsinized and seeded on f15 mm glass coverslips for immunocytochemistry or 6-well culture plates for other experiments. At this stage, approximately 95% of the cells showed immunoreactivity for glial fibrillary acidic protein (GFAP). Cultured astrocytes were treated with TPNA10168 (Maybridge, Loughborough, UK; Fig. 1a) and other reagents in serum-free MEM at 37 °C.
Measurement of mRNA levels:
Total RNA was extracted from cultured astrocytes using a total RNA extraction kit (Favorgen Biotech Corp., Ping-Tung, Taiwan). Reverse transcription and quantitative PCR were performed as previously described (Koyama et al. 2019). The following primer pairs were used:
5’-ACCATTGAAATACACATACACCCT-3’ and 5’-GTTGCAAACACCGAGCTTCC-3’;
g-GCLc, 5’-CATGAAAGTGGCCCAGAAGC-3’ and 5’-CCGCCTTTGCAGATGTCTTT-3’;
g-GCLm, 5’-CGAGGAGCTTCGGGACTGTA-3’ and 5’-CCCTGACTAAATCGGGGCTG-3’;
Nrf-2, 5’-CTCCTAGTTCTCGCTGCTCG-3’ and 5’-TGGCAACTCCAAGTCCATCA-3’;
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
5’-CTCATGACCACAGTCCATGC-3’ and 5’-TACATTGGGGGTAGGAACAC-3’.
Serial dilutions of each amplicon were amplified in the same manner as the standard for the copy number of PCR products. The amount of mRNA was calculated as the copy number of each reverse-transcription product (equivalent to 1 µg of total RNA) and normalized to the value of GAPDH.
Cell lysate preparation and SDS-PAGE were performed as previously described (Koyama et al. 2019). To detect Nrf-2, system Xc-, g-GCLc, and g-GCLm proteins, the membranes were first probed with an anti-Nrf-2 rabbit antibody (1:2,000 dilution, D1Z9C cat# 12721; Cell Signaling Technology, Danvers, MA, USA), anti-system Xc-/SLC7A11 rabbit antibody (1:2,000 dilution, cat# 98051; Cell Signaling Technology), anti-g-GCLc mouse antibody (1:2,000 dilution, clone H-5, cat# sc-390811; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or anti-g-GCLm mouse antibody (1:2,000 dilution, clone E-4, cat# sc-55586; Santa Cruz Biotechnology), followed by incubation with peroxidase-conjugated secondary antibodies. Labeled protein bands were detected using an enhanced chemiluminescence kit (Chemi-Lumi One L; Nacalai Tesque, Kyoto, Japan). The chemiluminescence of each band was observed using a Lumino Image Analyzer (ImageQuant LAS4000, GE Healthcare, Chicago, IL, USA), and the density of the target protein bands was quantified using ImageJ 1.45 software (US National Institutes of Health, Bethesda, MD, USA). The membranes were then stripped and re-probed with a mouse anti-β-actin primary antibody (1:10,000 dilution, cat# MAB1501; Millipore, Burlington, MA, USA). The results are presented as the ratio of the level of β-actin protein. Whole uncropped immunoblot images obtained using these antibodies are shown in Supplementary Fig. 1.
After treatment with TPNA10168, cultured astrocytes were fixed with 3% paraformaldehyde and incubated with a rabbit antibody against Nrf-2 (1:400 dilution, D1Z9C cat# 12721; Cell Signaling Technology). To identify astrocytes, an anti-glial fibrillary acidic protein (GFAP) mouse monoclonal antibody (1:400 dilution, G-A-5; Sigma, St. Louis, MO, USA) was used. After incubation with primary antibodies, the cells were labeled with FITC-conjugated anti-rabbit IgG and rhodamine-conjugated anti-mouse IgG. After incubation with secondary antibodies, 5 µg/mL Hoechst 33342 (cat# H341; Dojindo, Kumamoto, Japan) was used to label the nucleus. Labeled cells were observed under an epifluorescence microscope.
DNA binding activity of Nrf-2:
Nuclear extraction was performed using a nuclear extraction kit (cat# 40010; Active Motif, Carlsbad, CA, USA). After treatment with TNPA10168, the nuclear extract was prepared from cultured astrocytes according to the manufacturer’s protocol. The binding of Nrf-2 protein to the DNA consensus sequence was measured using an ELISA-based assay kit (TransAM® Nrf2 Transcription Factor Assay Kit, cat# 50296; Active Motif); 2 µg of protein from astrocytic nuclear extracts was used for each treatment. The DNA binding assay was performed according to the manufacturer’s protocol.
Measurement of intracellular total glutathione:
After treatment with TPNA10168 and/or rotenone, cultured astrocytes were collected in a 1.5 mL centrifuge tube with 300 µL of phosphate-buffered saline and centrifuged. The resulting cell pellets were lysed in 80 µL of 10 mM HCl containing 5-sulfosalicylic acid and centrifuged at 8000 × g for 10 min. The total glutathione content (sum of the reduced and oxidized forms) in the supernatant was measured using a glutathione quantification kit (Cat # T419; Dojindo). The resulting pellet was solubilized with 0.1 M NaOH, and the protein content was measured. The results are presented as glutathione content per mg protein.
After treatment with TPNA10168 and/or rotenone for 48 h, astrocytes were incubated with 1 µM calcein-AM and 1 µM ethidium homodimer III (EthD-III) (Live/Dead Cell Staining Kit II, cat# D25497; Takara Bio. Inc., Kusatsu, Japan) in serum-free MEM at room temperature for 30 min and observed under an epifluorescence microscope. Cells exhibiting calcein fluorescence in the cytoplasm and EtD-III fluorescence in the nucleus were defined as living and dead cells, respectively. In each condition, more than 400 cells were observed, and the percentage of dead cells was indicated by the ratio of the total cells observed (sum of living and dead cells).
All statistical analyses were performed using Bell Curve for Excel (version 2.20; Social Survey Research Information Co., Ltd, Tokyo, Japan). Experimental data are presented as the mean ± standard error of the mean (SEM). The results were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc test. Statistical significance was set at P < 0.05.