The paraffin sections of tumor tissues were dewaxed and dehydrated for antigen repair. The slides were incubated with anti-KI67 (1:150) at 4 °C overnight and washed with PBS. Then the sections were incubated with goat anti-rabbit IgG (1:2000) for 15 min followed by stained with DAB solution for 5 min. Finally, the sections were sealed by neutral resin and the images were captured by a microscope (DS-Ri2, Nikon).

The prussian blue (PB) staining (60533ES60, YEASEN) was employed to detect iron deposition in tumor tissues. Tumor tissues were fixed in Fixation Buffer and embedded in paraffin, followed by cutting in 4-µm slices. Deparaffinized sections were stained with Perls’ stain for 30 min, and Nuclear Fast Red solution for 5 min. The sections were sealed and observed under a microscope.

SKOV3 cells were seeded on sterilized coverslips and then fixed with 4% paraformaldehyde followed by permeabilized with 0.5% Triton X-100 for 15 min. The paraffin sections of tumor tissues were dewaxed and dehydrated for antigen repair. Then cells and sections were incubated with anti-LC3 (1:200, ab48394, Abcam), anti-ATG5 (1:200, ab108327, Abcam) or anti-NCOA4 (1:200, ab48394, Abcam) at 4 °C overnight, then were incubated with secondary antibody conjugated to fluorescein isothiocyanate for 1 h in the dark. Finally, cells were stained with DAPI and observed under a fluorescence microscope.

The tumor tissues were cut into small pieces and homogenized in PBS at 4 °C. Then subsequent centrifugation at 1500 x g for a duration of 20 min to obtain the supernatant. SKOV3 cells were centrifuged at 1000 x g for 10 min at 4˚C to collect the supernatant. The levels of 4-HNE, MDA, SOD and GSH-Px in the supernatant were measured by a ELISA assay kit (Nanjing Jiancheng).

The total proteins form SKOV3 cells and tumor tissues were extracted by RIPA lysis buffer (Beyotime) containing protease inhibitors, and quantified with BCA Protein Assay Kit (epizyme). 30 µg of protein sample s were separated by 12% SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated with primary antibodies at 4˚C overnight after blocking with 5% BSA and subsequently incubated with secondary antibody (ab6721, 1:5,000, Abcam) for 1 h. An Enhanced ECL Kit (Yeasen) was used to visualize the protein bands and the band intensity was measured with ImageJ software. The primary antibodies including: anti-LC3 (1:1000, ab192890, Abcam), anti-Beclin1 (1:1000, ab207612, Abcam), anti-p62 (1:1000, ab109012, Abcam), anti-GPX4 (1:1000, ab41787, Abcam), anti-ACSL4 (1:1500, ab155282, Abcam), anti-TFR1 (1:1500, AF5343, Affinity), anti-ATG5 (1:1000, ab108327, Abcam), anti-NCOA4 (1:1000, ab48394, Abcam) and anti-GAPDH (1:1000, ab8245, Abcam).

The data was analyzed by GraphPad Prism 8 software and showed in the format of mean ± standard deviation (SD). The unpaired student’s t-test was employed to compare the significance between two groups, a One-way ANOVA was performed accompanied by Tukey’s post hoc test was used to compare three or more groups. p < 0.05 means statistically significant difference.