Cell culture

KGN cell line (a human ovarian granulosa cell line, Procell CL-0603) was purchased from Procell Life Science&Technology Co., Ltd. (Wuhan, China). Cells were plated in a 6-cm dish (Corning, New York, USA) and cultured in DMEM/F12 (Hyclone, Logan, UT) medium supplemented with 10% FBS (Sijiqing, China) at 37 ℃ with 5% CO2 atmosphere. Cells were passaged every 2 days. According to our previous studies [18, 34], the concentration of cisplatin used in this study was 10 µM.

Western blot analysis

Cell proteins were extracted using radioimmunoprecipitation buffer (RIPA, 1 ×), containing 1% phenylmethylsulfonyl (PMSF) and 2% phosphorylase inhibitor (PI) on ice. BCA protein assay kit (Beyotime, Shanghai, China) was applied to measure the protein concentration. A total of 30 μg proteins per sample was separated on 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gels at 80 V for 30 min and 120 V for 1.5 h. Then, the proteins were transferred onto NC membranes (PALL, Germany) at 300 mA for 1 h. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated them at 4 ℃ overnight with the primary antibodies against GPX4 (1:1000, Abcam, ab125066, USA), Nrf2 (1:1000, Abcam, ab180845, USA), x-CT (1:1000, Abmart, T57046, China), FTO (1:1000, Abcam, ab126605, USA), ATF4 (1:1000, ab270980, Abcam, USA), ACSL4 (1:1000, Abmart, T510198, China), β-actin (1:2000, 66,099–1-Ig, Proteintech, China), and GAPDH (1:5000, AP0063, Bioworld Technology, USA). The next day, the membranes were washed with TBS containing 0.1% Tween‐20 (TBST) for 3 times and incubated in peroxidase-conjugated secondary antibodies goat antirabbit IgG (1:5000, zhuangzhishengwu, Xi’an, China) at room temperature for 1 h. Finally, visualize these membranes using a ECL Kit (Millipore, MA).

Measurement of reactive oxygen species (ROS) level

After granulosa cell being treated with cisplatin or injured granulosa cell being transfected plasmid or si-RNA for 48 h in a 6-well plate, the cells were harvested. According to the manufacturer’s instructions (S0033S, Beyotime, China), diluting 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) with the serum-free culture medium (DMEM/F12) at 1:1000 to a final concentration of 10 μM. Then, cells (10 × 104/sample) were resuspended with DCFH-DA and incubated at 37 ℃ for 15 min in a 1.5-mL EP tube. During this period, the EP tube was inverted every 3–5 min, allowing the probe and cells to mix evenly and come into full contact. Finally, the cells were washed with DMEM/F12 culture medium three times and the cells were transferred into a 96-well plate. The ROS production was measured at 488 nm excitation and 525 nm emission using a fluorescence spectrophotometer (BioTek).

Detection of intracellular glutathione peroxidase (GPx)

To measure the total intracellular GPx, the cellular glutathione peroxidase assay kit with NADPH (S0056, Beyotime, China) was applied. First, cells were collected using cell scrapers and the cells washed with PBS. The cells were lysed with cell lysis buffer (100 μL/100 × 104 cells) for 15 min on ice. After being centrifuged at 4 ℃ for 10 min, the supernatant was collected for the enzyme activity measurement. We prepared the GPx detection working solution, glutathione peroxidase detection buffer, sample, and 30 mM peroxide reagent solution. Then, according to the instructions, after 30 mM peroxide reagent solution was added, mixed immediately, and transferred them to a 96-well plate. The activity of GPx was measured using a fluorescence spectrophotometer (BioTek) at 340 nm at room temperature.

Measurement of glutathione (GSH)

To detect the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG), the GSH and GSSG Assay Kit (S0053, Beyotime, China) was applied. We first detected the content of total glutathione in the sample. Briefly, cells were washed with PBS, centrifuged, and collected the precipitates. Add a protein removal reagent M solution that is 3 times the volume of the cell precipitate and fully vortex. Then, the sample was subjected to two rapid freeze-thaws using liquid nitrogen and a 37 ℃ water bath. After being placed on ice for 5 min, centrifuge the sample at 10,000 g at 4 ℃ for 10 min. The supernatant was collected for the determination of total glutathione. Next, take some of the above prepared samples to test the content of GSSG. Add 20 μL of diluted GSH removal auxiliary solution and 4 μL of GSH removal reagent working solution to every 100 μL of the sample, mix them with vortex immediately, and react for 60 min at room temperature. Finally, transfer the above prepared samples to a 96-well plate and measure the absorbance at 412 nm using a fluorescence spectrophotometer (BioTek) at 25 ℃. The GSH content can be subsequently calculated according to the manufacturer’s instructions.

Detection of malondialdehyde (MDA)

Higher intracellular malondialdehyde (MDA) levels suggest lipid peroxidation. To detect the level of injured granulosa intracellular MDA, the lipid peroxidation MDA assay kit was applied (S0131S, Beyotime, China). Cells (100 × 104) were lysed using 100 μL RIPA lysate for 30 min on ice, centrifuged at 12,000 g for 10 min, and collected the supernatant to for the subsequent experiment. Add 100 μL sample and 200 μL MDA working solution to a 1.5-mL EP tube, fully mix, and heat at 100 ℃ for 15 min. After being cooled to room temperature, centrifuge 1000 g at 25 ℃ for 10 min. Then, transfer 200 μL of supernatant to a 96-well plate and measure the absorbance at 532 nm using a fluorescence spectrophotometer (BioTek) at 25 °C.

Transmission electron microscopy (TEM) assay

To observe the morphological change of cisplatin on granulosa cell mitochondria, TEM assay was applied. First, fix the cell samples with 2% glutaraldehyde‐paraformaldehyde in 0.1 M Na‐phosphate buffer (PB) for 2 h, and wash the samples with 0.1 M PB three times for 15 min. Then, fix the samples 1% OsO4 dissolved in 0.1 M PB for 2.5 h and wash them with 0.1 M PB three times for 15 min. Dehydrate the samples in an ascending gradual series of ethanol at 4 ℃ for 15–20 min, respectively. Treat the samples with 100% acetone three times for 15–20 min at room temperature. After embedding the sample with pure embedding solution at 37 ℃ for 2 h, the samples were sequentially polymerized at 37 ℃ overnight and at 45 ℃ and 60 ℃ for 24 h. Slice the samples with LKB microtome with a thickness of about 60 nm. Finally, double stain the samples with 3% uranyl acetate and lead citrate and visualize them with a transmission electron microscope (Japan, HITACHI, H-7650).

Cell proliferation assay

1 × 104 transfected cells/well were plated in a 96-well plate and cultured at 37 ℃ with 5% CO2 overnight. Then, 10 μL CCK-8 reagent and 90 μL complete culture medium were added to each well. After being cultured 2 to 4 h, the absorbance was detected at 450 nm.

Transfection

25 × 104 cells/per well were planted in a 6-well plate and culture overnight. Next day, cell confluence reached 60–80% and transfected with pCAG-FTO (Miaoling, Wuhan, China) plasmid to promote the FTO expression, a non-specific sequence was used as a negative control (NC). Small interfering RNAs (siRNAs) against FTO (Ribobio, Guangzhou, China) and its negative control (si-NC) were used to downregulate the expression of FTO. For the ATF4, the pcDNA-ATF4 vector, DNA plasmids against ATF4 (sh-ATF4-1, sh-ATF4-2, sh-ATF4-3), and their negative control (GeneChem Co. Ltd, Shanghai, China) were also included. Transfection was conducted using the Lipofectamine 2000 according to the manufacturer’s instructions. Cells were harvested after treatment for 48 h, and the western blotting was applied to test the efficiency of transfection.

Statistical analysis

All analyses were carried out using the GraphPad Prism software 5.0. The results are shown as the mean ± SEM. Every experiment is performed in triplicate. Student’s t-test and one-way ANOVA are used to assess the differences between groups. P < 0.05 was considered significant. P values were shown in the figures as follows: ***P < 0.001, **P < 0.01, and *P < 0.05; n.s. indicated not significant.