Df6701

H9c2 cells (1 × 10⁵ cells/mL) were seeded into confocal laser dishes. Following dioscin (200 nM) pretreatment for 24 h and DOX (4 μM) intervention for 24 h, H9c2 cells were fixed with fresh 4% paraformaldehyde for 20 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100 for 15 min and blocked with 5% goat serum for 30 min. Antibodies against Nrf2 (A21176, ABclonal), NOX4 (DF6924, Affinity), and GPX4 (DF6701, Affinity) (antibody dilution ratio 1: 200) were incubated overnight at 4 °C, respectively. The cells were then incubated with the DyLight 488 Conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (1:200, BA1227, Boster) at room temperature for one hour. Similarly, cardiac tissue sections were permeabilized with a blocking solution containing 0.2% Triton X-100 and 5% goat serum for 30 min. Then, cardiac tissue sections were treated overnight with Nrf2 (A21176, ABclonal), NOX4 (DF6924, Affinity), and GPX4 (DF6701, Affinity) antibodies (antibody dilution ratio 1: 100), respectively, followed by incubation with DyLight 550 Conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (1:200, BA1135, Boster). Fluorescence changes were captured using confocal laser microscopy and quantified using ImageJ software version 2.0.

Paraffin-embedded renal tissue samples were sliced into 5-μm-thick sections, which were deparaffinized and rehydrated in a graded ethanol series. Antigen retrieval was achieved by boiling sections in antigen recovery buffer (10 min), followed by incubation in goat serum (15 min). Sections were then incubated with primary antibodies against GPX4 (1:200, DF6701, Affinity, China) and SLC7A11 (1:200, NB300-317SS, NOVUS, USA) (4 °C, overnight), rinsed three times, and immersed in Cy3-conjuated goat anti-rabbit IgG (1:200, A0516, Beyotime, China) (60 min, room temperature, in darkness). Cell nuclei were stained with DAPI (D106471-5 mg, Aladdin, China) and images captured using a fluorescence microscope (magnification, 400×).

We performed Western blot, as previously recorded ((Zhang et al., 2016 (link); Fei et al., 2020 (link))). The proteins extracted from renal tissues or cells were lysed in a RIPA buffer containing protease and phosphatase inhibitors, and the protein content was detected by using the BCA protein assay kit (Beyotime). Protein samples (50 ug) from each group were electrophoresed in 10% SDS-PAGE gel and then transferred to a PVDF membrane. After blocking with 5% nonfat dry milk, the blots were incubated with the primary antibodies against GPX4 (1: 1000, DF6701, Affinity), ACSL4 (1: 1000, A14439, Ablonal), Nrf2 (1: 1000, AF7904, Affinity), SIRT-1 (1: 1000, DF6033,Affinity), and or β-actin (1: 200, ab181602, Abcam) overnight at 4°C. Subsequently, the blots were washed in TBST and incubated with a secondary antibody for 2 h at room temperature. The bands were determined with an ECL system applying an ECL kit (Applygen), and band intensities were checked by BandScan software.