Hoechst staining solution

HepG2 cells were cultured and seeded on 24-well plates containing a sterile glass slide, with 20,000 cells per well. HepG2 cells were incubated with 1640 single medium containing 10, 20, or 30 µM sarmentosin for 12 h. Then, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 m at room temperature. The glass slide containing the cells was removed, and Hoechst staining solution (Beyotime, #C1017) was added dropwise and incubated for 20 m. Then, the mounting slide was quenched with anti-fluorescence. Glass slides with HepG2 cells were covered on glass slides and protected from light. A fluorescence microscope (Leica, DMI3000B) was used to take pictures.

HPDE6C7 cells were cultured with CAE for 48 h at 37°C and washed with PBS three times. Subsequently, 1 ml cell staining buffer was added, followed by 5 µl Hoechst staining solution and 5 µl propidium iodide (PI) staining solution (Beyotime Institute of Biotechnology) at the same time and placed at 4°C for 30 min in the dark. After washing with PBS three times, the dye mixture was discarded and the cells were observed under a fluorescence microscope.

R28 cells were seeded into 12-well culture plates (1 × 10⁵ cells/well) and divided into four groups: the control group, glutamate group (10 mM glutamate), SB202190 group (10 mM glutamate + 25 µM SB202190) and ferrostatin-1 group (glutamate 10 mM + 1 µM ferrostatin-1 [Ape Bio]). Glutamate was dissolved in cell culture medium to a concentration of 10 mM. ferrostatin-1 and SB202190 were dissolved in DMSO (MP Biomedicals) and diluted in culture medium containing 10 mM glutamate. Cells were treated with the respective treatments for 24 hours.
For propidium iodide (PI)/Hoechst 33342 staining, cells were incubated with cell staining buffer, Hoechst staining solution, and PI staining solution at 4°C in the dark for 30 minutes following the manufacturer’s instructions (Beyotime, Shanghai, China). Cells were washed with PBS and observed using a fluorescence microscope (Nikon, Tokyo, Japan). Propidium iodide (PI) indicated dead cells (red fluorescence) and Hoechst 33342 stained all cells (blue fluorescence). The brightness parameters were consistent between the groups during image capture. The photos were analyzed using ImageJ software (Fiji; National Institutes of Health, Bethesda, MD, USA; Schneider et al., 2012). The percentage of cell death was calculated using the formula: cell death rate% = PI-positive cells/Hoechst-positive cells × 100.

The primary antibodies of MITF, GAPDH, TYR, TYRP1, DCT, ras-related protein Rab-27A (RAB27A), nuclear factor E2-related factor 2 (NRF2), catalase (CAT), and heme oxygenase 1 (HO-1) were purchased from Cell Signaling Technology. The primary antibodies for BCL2 and BAX were from ZEN-BIOSCIENCE. Fraxin was purchased from Chemface (Hubei, China) and was stored at −20 °C after dissolving in dimethyl sulphoxide (DMSO) at 320 mM concentration. ML385 was purchased from Adooq Bioscience. Hydrogen peroxide (H₂O₂), DMSO, and Cell Counting Kit-8(CCK8) were procured from Biosharp (Hefei, China). The Fontana-Masson Stain Kit was purchased from G-CLONE. Hoechst staining solution (100 × ) was purchased from Beyotime Company.