Dcfh da

The Intracellular ROS levels were detected by DCFH-DA (Beyotime Biotechnology, S0033S). The Intracellular mitochondrial ROS levels were detected by Dihydrorhodamine 123 (MCE, HY-101894). Cells were cultured in six-well plates at a density of 2.0 × 10⁵ cells/well. After being treated with l-sorbose, the cells were gently washed with D-PBS (BBI, E607009) followed by incubation with DCFH-DA or Dihydrorhodamine 123 at 37 °C for 30 min.
To obtain microscopy images, cells were seeded on a confocal dish overnight and treated with l-sorbose for 6 h. After being treated with l-sorbose, the cells were gently washed with D-PBS followed by incubation with MitoBright LT (Dojindo Laboratories, MT11) and DCFH-DA (Dojindo Laboratories, CK04) at 37 °C for 30 min, or Dihydrorhodamine 123 alone. Then the cells were washed with D-PBS twice. Images were obtained with a Nikon C2 Eclipse Ti-E inverted confocal microscope equipped with NIS-Element AR software.
To measure the volume of mitochondria, cells were cultured in six-well plates at a density of 2.0 × 10⁵ cells/well. After being treated with l-sorbose, the cells were gently washed with D-PBS followed by incubation with MitoBright at 37 °C for 30 min. Then the cells were washed twice with D-PBS. The fluorescence of the cells was measured immediately on a FACSCalibur flow cytometer and analyzed by FlowJo software.

The levels of intracellular ROS were evaluated using the peroxide-sensitive fluorescent probe 2′7’-dichlorofluorescein diacetate (DCFH-DA) (Dojindo, Kumamoto, Japan). The sperm were exposed to a serum-free medium containing 10 μM DCFH-DA in the dark for 30 min and then washed thrice with cold PBS. Flow cytometry was used to measure the fluorescence intensity (FACS Calibur, Becton-Dickinson, Sunnyvale, CA).

Donafenib was provided by Suzhou Zelgen Biopharmaceuticals Co, Ltd.; GSK‐J4 (HY‐15648B), sorafenib (HY‐10201A), regorafenib (HY‐10331), Z‐VAD‐FMK (HY‐16658), necrosulfonamide‐1 (HY‐100573), 3‐MA (HY‐19312), hemin (HY‐19424), BSO(HY‐106376), Tocopherol(HY‐131553), DFO(HY‐B0988) and zinc protoporphyrin (ZnPP; HY‐101193) were purchased from MCE; Fer‐1 (S7243) was purchased from Selleck; CCK‐8(C0005) was purchased from Targetmol; Liperfluo(L248), MDA Assay Kit(M496) and DCFH‐DA(R253) was purchased from Dojindo; CellTiter‐Glo® 3D Cell Viability Assay was purchased from Promega(G9681), propidium iodide (PI) was purchased from Beyotime; and FeRhoNoxTM‐1(GC901) was purchased from Goryo. Antibodies against KDM6A (ab253183), GPC3 (ab207080), NRF2 (ab62352), H3K4me1 (ab8895) and H3K27ac (ab4729) were purchased from Abcam; antibody against HA‐tag (HT301) was purchased from TransGen Biotech, antibodies against HMOX1 (10701‐1‐AP) and Actin (66009‐1‐Ig) and were purchased from Proteingroup; and antibody against H3K27me3 (07‐449) was purchased from Sigma. ATAC kit (N248) and CUT&Tag kit (N259) were purchased from novoprotein.

Intracellular ROS levels were measured using a photo-oxidation-resistant DCFH-DA assay according to the manufacturer's instructions (#R253, Dojindo, Japan). HeLa cells were harvested using the same method mentioned in “Annexin A5 Apoptosis Detection assay.” The harvested cells were then washed with fresh medium and incubated with DCFH-DA at a final concentration of 10 nM in the loading buffer provided in the kit. After incubation for 30 min at 37 °C, the cells were washed thrice with the medium. The ROS levels were analyzed using an RF-500 flow cytometer, and the data were analyzed using FCSalyzer 0.9.22 (https://sourceforge.net/projects/fcsalyzer/).

The cells were seeded in 24‐well plates and after being treated with 9‐PAHSA or S‐9‐PAHSA (60 μM) for 24 h, the medium was removed and the cells were washed twice with PBS, followed by the addition of DCFH‐DA (Dojindo Laboratories, Japan) and incubation for 30 min in the dark at 37°C with 5% CO₂. After culturing, the cells were washed three times with a serum‐free medium, followed by the addition of fresh medium and detection of the ROS level using a fluorescent microscope. The intensity of fluorescence was calculated using Image J (National Institutes of Health, USA).