RNA extraction

EC cells were cultured to 80–90% confluency and then digested with 0.25% trypsin–EDTA (25,200,056, Thermo Fisher, USA) for 3–5 min. Digestion was halted with a culture medium containing 10% FBS (10100147C, Thermo Fisher, USA). Cells were collected by centrifugation at 1000 rpm for 5 min, and the supernatant was discarded. Total RNA was extracted using the Trizol reagent kit (A33254, Thermo Fisher, USA) according to the manufacturer's instructions. The RNA precipitate was washed with 75% ethanol (E299585, Aladdin, Shanghai, China), air-dried for 5–10 min, and dissolved in DEPC-treated water (Akbar et al. 2019). The purpose of RNA extraction was to obtain high-quality RNA for subsequent high-throughput transcriptome sequencing.

The purity and integrity of RNA were assessed using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher) by measuring the OD260/280 ratio, ensuring no protein or organic contamination. RNA concentration was measured with the Qubit RNA assay kit (Q33221, Thermo Fisher, USA). RNA samples meeting the criteria of RNA integrity number (RIN) ≥ 7.0, 28S:18S ratio ≥ 1.5 were used for further experiments (Gonye et al. 2023).

High-throughput transcriptome sequencing

Sequencing libraries were prepared and sequenced by CapitalBio Technology (Beijing, China) using 5 μg of RNA per sample. Ribosomal RNA (rRNA) was removed from total RNA using the Ribo-Zero Magnetic Kit (MRZG12324, Epicentre, USA). The Illumina NEB Next Ultra RNA Library Prep Kit (E7760S, NEB, USA) was used for library construction. RNA fragments were then fragmented into approximately 300 base pair (bp) segments in NEB Next First Strand Synthesis Reaction Buffer (5×). First-strand cDNA was synthesized using reverse transcriptase and random primers, followed by second-strand cDNA synthesis in dUTP Mix (10×) Second Strand Synthesis Reaction Buffer. The ends of the cDNA fragments were repaired, including the addition of A-tails and adapter ligation. After ligating the Illumina sequencing adapters, the second strand cDNA was digested using the USER enzyme (M5508, NEB, USA) to construct a strand-specific library. The library DNA was amplified, purified, and enriched by PCR. The library was validated using an Agilent 2100 instrument and quantified with the KAPA Library Quantification Kit (kk3605, Merck, USA). Finally, paired-end sequencing was performed on the Illumina NextSeqCN500 platform (Ayturk 2019; Simoneau et al. 2021). This experiment was undertaken to identify differentially expressed genes (DEGs), and the results guided subsequent functional analyses.

Transcriptome sequencing data analysis

The quality of the raw sequencing paired-end reads was assessed using FastQC software v0.11.8. The initial data were preprocessed with Cutadapt software v1.18 to remove Illumina sequencing adapters and poly(A) tails. Reads containing more than 5% ambiguous bases (N) were discarded using Perl scripts. The FASTX Toolkit v0.0.13 was employed to retain reads, with at least 70% of bases having a quality score above 20. BBMap software was used to repair paired-end sequences. Finally, high-quality filtered reads were aligned to the human reference genome using hisat2 software v0.7.12. This preprocessing step ensured the data quality for subsequent differential expression analysis.

Differential expression analysis of mRNA read counts was conducted using the "edgeR" package in R, with criteria set at |log2FC|> 1 and p < 0.05. This step aimed to identify genes significantly associated with EC progression and provided a basis for further functional analyses.

Autophagy-related genes were identified from the GeneCards database (https://www.genecards.org/) using the search term "Autophagy," resulting in 327 relevant genes. A Venn analysis was performed using the "VennDiagram" package in R to intersect these genes with the DEGs identified earlier. This intersection yielded significant autophagy-related DEGs (Stelzer et al. 2016).

A heatmap of the intersecting genes was generated using the "heatmap" package in R. Protein–protein interaction (PPI) analysis of key factors was performed using the STRING database (https://string-db.org/) with a minimum interaction score of 0.700. The interaction networks were visualized with Cytoscape v3.5.1, and core genes were identified using the CytoHubba tool.

The "ClusterProfiler" package in R was used for Gene Ontology (GO) functional enrichment analysis of the intersecting genes, covering biological processes (BP), molecular functions (MF), and cellular components (CC). Results were visualized with bubble and circle plots, using P < 0.05 as the threshold. Additionally, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on candidate targets using the "ClusterProfiler" package, with visualizations in bubble plots and circle plots (Shi et al. 2020).

TCGA data analysis for survival analysis

RNA-seq data (FPKM format) and clinical data for the TCGA-UCEC (Uterine Corpus EC) project (n = 553) were downloaded from the TCGA database (https://portal.gdc.cancer.gov). Samples lacking clinical information or normal samples were excluded. Proportional hazard assumptions were tested, and survival regressions were fitted using the "survival" package in R. The results were visualized with the "survminer" and "ggplot2" packages in R (Liu et al. 2018). This survival analysis aimed to determine the prognostic significance of SIRT1 and FOXO3 in EC, further supporting their potential as therapeutic targets.

Cell culture and in vitro experimental protocols

The human embryonic kidney cells (HEK-293T) and EC cells (Ishikawa, RL95-2, KLE, AN3CA, and HHUA) were all purchased from Biobw Biotechnology Co., Ltd. (Bio-72947, Bio-73224, Bio-73138, Bio-73074, Bio-53662, and Bio-133241; Beijing, China). Human endometrial cells (EEC) were obtained from KeyCell Biotechnology Co., Ltd. (QS-H011, Wuhan, China) and cultured in MEM medium supplemented with non-essential amino acids (NEAA) (PM150410, Procell Co., Ltd.; Wuhan, China) containing 10% FBS and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, 15,140,163, Thermo Fisher, USA). The remaining cell lines were cultured in high-glucose DMEM medium (11,965,084, Thermo Fisher, USA) supplemented with 10% FBS and 1% antibiotics. All cells were maintained in a humidified CO2 incubator (Heracell™ Vios 160i CR CO2 incubator, 51,033,770, Thermo Fisher) at 37 °C with 5% CO2. Cells were passaged when they reached 80–90% confluency (Asaka et al. 2015).

For cell treatments, cells were seeded in 6-well plates and allowed to adhere overnight. Then, a deacetylase inhibitor cocktail (DIC) (P1112, Beyotime, Shanghai, China) was added to the cell culture medium at a 1:100 ratio following the manufacturer's instructions for 24 h of treatment (Zheng et al. 2022). Alternatively, cells were treated with 10 μM of the autophagy inhibitor chloroquine (CQ, HY-17589A, MedChemExpress, USA) for 24 h before subsequent experiments (Xu et al. 2024). These treatments aimed to investigate the effects of deacetylase inhibition and autophagy inhibition on EC cells.

For cell grouping, cells were divided into the following groups to explore the interactions between SIRT1 and FOXO3 and their effects on EC cell behavior: negative control (NC) of short hairpin RNA (shRNA, sh-NC), shRNA targeting SIRT (sh-SIRT1), overexpression (oe)-NC, oe-SIRT1, sh-NC, sh-FOXO3, oe-NC + dimethyl sulfoxide (DMSO), oe-NC + DIC, oe-SIRT1 + DMSO, oe-SIRT1 + DIC, oe-SIRT1 + DMSO, oe-SIRT1 + CQ, oe-NC + sh-NC, oe-SIRT1 + sh-NC, oe-SIRT1 + sh-FOXO3.

Lentivirus and plasmid transduction

Lentiviral infection was employed to overexpress or silence genes in EC cells, with lentiviral packaging services provided by Sangon Biotech, Shanghai, China. Plasmids from the pHAGE-puro series and auxiliary plasmids pSPAX2 and pMD2.G (Addgene, USA, catalog numbers #118,692, #12,260, and #12,259) were co-transfected into HEK293T cells along with pSuper-retro-puro series plasmids and auxiliary plasmids gag/pol and VSVG (catalog numbers #113,535, #14,887, and #8454). After 48 h of cell culture, the supernatant containing lentiviral particles was collected and filtered through a 0.45 μm filter. A second collection was performed at 72 h, and the viral particles were concentrated by centrifugation. The two viral harvests were combined, and viral titers were determined.

The logarithmic-phase cells were digested with trypsin and seeded at a density of 1 × 105 cells per well in a 6-well plate. After 24 h of routine culture, when the cell confluence reached approximately 75%, cells were infected with lentiviral particles (MOI = 10, working titer approximately 5 × 106 TU/mL) and 5 μg/mL polybrene (TR-1003, Merck, USA). After 4 h, the medium was diluted with an equal volume of culture medium to reduce polybrene concentration. The medium was replaced with fresh culture medium 24 h post-infection.

Cells were selected with puromycin (E607054, Sangon Biotech) to construct stably transfected cell lines at an appropriate concentration. Lentiviral silencing sequences are shown in Table S1, and the sequences with the best silencing efficiency were used for subsequent experiments.

RT-qPCR

Total RNA from cells was extracted using the Trizol reagent kit, and cDNA was synthesized using the PrimeScript RT Reagent Kit (RR047A, Takara, Japan). RT-qPCR was performed using the SYBR® Premix Ex TaqTM II kit (DRR081, Takara, Japan) on an ABI7500 real-time PCR system (Thermo Fisher, USA). GAPDH was used as the internal control, and each RT-qPCR experiment included three technical replicates. The relative expression of target genes was calculated using the 2−ΔΔCt formula (Ayuk et al. 2016). Primer sequences are detailed in Table S2.

Western blot

Cells were lysed using RIPA lysis buffer containing 1% PMSF (P0013B, Beyotime) to extract total protein, following the manufacturer's instructions. The total protein concentration of each sample was determined using a BCA Protein Assay Kit (P0011, Beyotime) and adjusted to 1 μg/μL. Based on the target protein size, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 8–12%) was prepared. Next, 50 μg of protein samples were loaded into each lane. The proteins on the gel were then transferred onto a PVDF membrane (1,620,177, Bio-Rad, USA). The membrane was blocked with 5% non-fat milk in 1 × TBST at room temperature for 1 h. The membrane was incubated overnight at 4 °C with the primary antibodies (antibody information provided in Table S3). Subsequently, the membrane was then incubated at room temperature for 1 h with HRP-conjugated goat anti-rabbit IgG (ab6721, Abcam, Cambridge, UK, 1:5000) or goat anti-mouse IgG (ab205719, Abcam, 1:5000) secondary antibody. The blots were visualized in ECL reaction solution (1,705,062, Bio-Rad, USA), and the bands were imaged using an Image Quant LAS 4000C gel imaging system (GE, USA). The grayscale values of the target bands were normalized to the internal reference band GAPDH (Walentowicz-Sadlecka et al. 2019).

Co-immunoprecipitation (Co-IP)

The Co-IP experiment was performed to detect PPIs in RL95-2 cells, particularly between SIRT1 and FOXO3. RL95-2 cells were lysed on ice for 10 min using an IP lysis buffer containing protease and phosphatase inhibitors (P0013, Beyotime). Lysates were centrifuged at 12,000g for 20 min at 4 °C, and the supernatant was collected. Twenty microliters of the lysate were set aside as input, and the remainder was incubated with 10 µL of Protein G magnetic beads (10004D, Thermo Fisher, USA) and 1 µL of anti-SIRT1 (2493S, Cell Signal Technology, USA) or anti-FOXO3 (12829S, Cell Signal Technology, USA) antibodies. The mixture was incubated on a shaker overnight at 4 ℃. The immunocomplexes were then washed four times with NETN buffer NETN buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40), separated by SDS-PAGE, and analyzed by Western blot using appropriate antibodies (Che et al. 2020).

SIRT1 activity assay

The SIRT1 enzyme activity was measured using the SIRT1 enzyme activity assay kit (ab156065, Abcam) according to the manufacturer's instructions. In each well of a microplate, 25 µL of HPLC-grade H2O, 5 µL of SIRT1 detection buffer, 5 µL of fluorescent substrate peptide, and 5 µL of NAD were added. Then, 5 µL of the sample was added to each well. The reaction was initiated by adding 5 µL of developing reagent to each well at 24.0 ± 2.0 ℃, followed by thorough mixing. The fluorescence intensity was measured using a microplate fluorometer (SpectraMax M3, USA) with an excitation wavelength of 360 nm and an emission wavelength of 485 nm. The measurements were taken every 2 min for a total duration of 60 min. The SIRT1 enzyme activity of the extract was calculated using the following formula (Salee et al. 2022):

$$SIRT1 activity=(\frac_}_})\times 100\%$$

FIsample and FIcontrol represented the fluorescence intensities of the sample and the control solution, respectively. The enzymatic activity of SIRT1 was expressed as the ratio of fluorescence intensity (Salee et al. 2022). This assay was used to assess the functional activity of SIRT1 in EC cells and delineate its role in mitophagy and hormone resistance.

CCK-8 cell viability assay

Cell concentration was adjusted to 1 × 103 cells/mL and then seeded into a 96-well plate with a volume of 100 μL per well. Cell viability was assessed at 12, 24, 36, and 48 h using the CCK-8 kit (C0041, Beyotime), following the manufacturer’s instructions. Subsequently, 10 μL of CCK-8 solution was added to each well and incubated at 37 ℃ and 5% CO2 for 2 h. The absorbance at 450 nm was measured using an ELISA reader to calculate cell viability (Liu et al. 2021).

EdU staining assay

EC cells were seeded in 24-well plates at a density of 1 × 105 cells per well, with three replicates per group. EdU solution (ST067, Beyotime) was added to the medium at a final concentration of 10 µmol/L and incubated for 2 h. Cells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed twice with PBS containing 3% BSA, and permeabilized with 0.5% Triton-100 in PBS for 20 min. Next, 100 µL of staining solution was added to each well, and the cells were incubated at room temperature in the dark for 30 min. Then, DAPI (C1002, Beyotime) was added to stain the cell nuclei for 5 min. The percentage of EdU-positive cells was calculated under a fluorescence microscope (FM-600, Shanghai Putian Optical Instrument Co., Ltd.) by observing 6–10 random fields per well (Yu et al. 2020). This assay was used to asses cell proliferation and determine the effects of gene modulation on cell cycle progression.

Colony formation assay

EC cells were seeded into each well of a six-well plate and cultured for 2 weeks, with the medium changed every 3 days. Colonies were fixed with methanol for 20 min and stained with 0.1% crystal violet (C0121, Beyotime) for 15 min. After rinsing, colonies were photographed and analyzed using Image Pro Plus 6.0 software (Liu et al. 2021). This assay provided insights into the clonogenic potential of treated cells and informed further investigations.

Transwell assay

Transwell invasion assays were performed after 24 h of different treatments. Transwell chambers were coated with 50 µL of Matrigel (354,234, BD Biosciences, USA) and incubated at 37 °C for 30 min to solidify. Cells were diluted to 2.5 × 104 cells/mL, and 100 µL of cell suspension was added to the upper chamber, while 500 µL of medium containing 10% FBS was added to the lower chamber. After 24 h, the cells on the upper surface of the membrane were removed with a cotton swab, and the cells that had invaded through the membrane were fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet for 30 min, and photographed under an inverted microscope (IXplore Pro, Olympus, Japan). Five random fields were counted for each well (Fan et al. 2020). The migration assay followed the same steps but without Matrigel coating. These assays were performed to evaluate the invasive and migratory abilities of treated cells.

Wound healing assay

Lines were drawn at the bottom of a six-well plate using a ruler and marker at intervals of 0.5–1 cm, with at least five lines per well. EC cells were seeded at a density of 5 × 105 cells per well. When the cells reached 100% confluency, scratches were made perpendicular to the drawn lines using a 200 µL pipette tip. The medium was replaced with serum-free medium, and images of the scratches were captured at 0 and 24 h using an optical microscope (DM500, Leica). The distance between the wound edges was measured using ImageJ software, and the wound healing rate was calculated using the following formula (Liu et al. 2021):

$$Wound healing rate=\frac_-distance}_}_}$$

"distance0 h" and "distance24 h" represent the distances between the cell scratches at 0 h and 24 h after scratching, respectively. This assay was conducted to evaluate cell migration and inform further studies on cell motility.

Flow cytometry

Apoptosis in EC cells was detected using the Annexin V-FITC/PI apoptosis detection kit (C1062L, Beyotime). Cells were seeded at 1 × 106 cells per well in six-well plates. After treatment, cells were collected, resuspended in 195 µL Annexin V-FITC binding buffer, and incubated with 5 µL Annexin V-FITC and 10 µL PI solution for 15 min in the dark. Flow cytometry analysis was performed within 20 min using a BD FACS Calibur flow cytometer to determine apoptosis rates (Liu et al. 2021).

Mitochondrial membrane potential (MMP) was assessed using the JC-1 kit (KGA604, Jiangsu KeyGen Biotech, China) according to the manufacturer's instructions. Briefly, 1 × 106 EC cells were resuspended in 500 μL of JC-1 staining working solution and incubated at 37 °C for 20 min. Cells were centrifuged at 2000 rpm for 5 min and resuspended in 500 μL of 1 × Incubation buffer, and analyzed by flow cytometry (excitation: 488 nm, emission: 530 nm) (Yao et al. 2022). These assays were adopted to elucidate the impact of treatments on cell apoptosis and mitochondrial function.

Transmission electron microscopy (TEM)

To observe EC cells under TEM, cells were first fixed in 3% glutaraldehyde (49,629, Sigma, USA) in 0.1 M phosphate buffer (pH 7.4, 17,202, Sigma, USA), followed by post-fixation with 1% osmium tetroxide (OsO4, O5500, Sigma, USA). After dehydration, 10 nm thick sections were prepared and stained with uranyl acetate (SPI-02624, Beijing Haide Chuangye Biotechnology, China) and lead nitrate (NIST928, Sigma, USA). Images were captured at 80 kV using a Hitachi H7650 TEM (Hitachi, Japan). Five random fields were selected for quantitative analysis of autophagic vacuoles (Lin et al. 2020). This step was carried out to visually confirm the presence of autophagic structures.

GFP-LC3B plasmid transfection

RL95-2 cells (1 × 105 cells per well) were seeded in a 24-well plate, with three replicates per group. After overnight incubation for cell adhesion, the GFP-LC3B plasmid (D2815, Beyotime) was transfected into RL95-2 cells using lentivirus. Following a 12-h transfection period, cells were fixed with 4% paraformaldehyde (P885233, Macklin, Shanghai, China) for 10 min. Cells were then stained with DAPI (#4083, Cell Signaling Technology, USA) for 10 min and mounted with 20 μL of mounting medium. Green fluorescent puncta were observed under a fluorescence microscope (Zeiss Observer Z1, Germany) (Chen et al. 2022). This experiment was conducted to monitor autophagosome formation.

Mitochondrial reactive oxygen species (ROS) detection

EC cells were digested, resuspended, and adjusted to a concentration of 1 × 106 cells/mL. Then, cells were plated (2 mL/well) into six-well plates and cultured overnight. Cells or tissues were washed with PBS and incubated with 5 μL of MitoSOX mitochondrial superoxide indicator (M36008, Thermo Fisher, USA) in PBS at 37 ℃ in the dark for 10 min. After washing with PBS to remove excess MitoSOX reagent, cells or tissues were fixed with 4% paraformaldehyde and stained with DAPI for 10 min. After three washes with PBS, cells were mounted with 20 μL of mounting medium and examined using a fluorescence microscope (Robinson et al. 2008). This assay was employed to assess mitochondrial ROS levels, informing further studies on oxidative stress in EC cells.

Immunofluorescence co-localization for Mitophagy

EC cells were digested, resuspended, and adjusted to a concentration of 1 × 106 cells/mL. Then, cells were plated (2 mL/well) into six-well plates and cultured overnight. After removing the culture medium, cells were washed twice with PBS. Mitochondrial selective probe Mitotracker Green FM (M7514, Thermo Fisher, USA) and lysosomal selective probe Lysotracker® Red DND-99 (L7528, Thermo Fisher, USA) were added to the cells at final concentrations of 0.1 µM and 0.025 µM, respectively, and incubated in the dark for 30 min. Cells were fixed with 4% paraformaldehyde and stained with DAPI for 10 min. Then, cells were mounted with 20 µL of mounting medium and examined using a fluorescence microscope (Shida et al. 2016; Li et al. 2017). This experiment was designed to visualize and quantify mitophagy by co-localizing mitochondria and lysosomes.

Cell immunofluorescence

Cells were fixed with 4% paraformaldehyde for 15–30 min and permeabilized with 0.1% Triton (L885651, Macklin) for 15 min. Next, cells were blocked with PBS containing 15% FBS at 5 °C overnight.

Cells were incubated with rabbit anti-FOXO3 antibody (MA5-14,932, 1:200, Thermo Fisher, USA) at 37 °C for 60 min. Subsequently, cells were incubated with FITC-conjugated goat anti-rabbit secondary antibody at 37 °C in the dark for 60 min. Finally, cells were stained with Alexa-488 conjugated goat anti-rabbit antibody (ab150129, Abcam) and DAPI for 1 h at room temperature. Cells were mounted with 20 μL of mounting medium for observation under a fluorescence microscope (Wagle et al. 2016). This experiment was carried out to determine the nuclear localization of FOXO3.

Furthermore, PINK1 and Parkin co-localization staining was conducted to study the interaction between PINK1 and Parkin in mitophagy. Cells were incubated with rabbit anti-PINK1 (ab216144, 1:500, Abcam) and anti-Parkin (A0968, 1:100, Abclonal, Wuhan, China) antibodies at 37 °C for 60 min. Next, cells were incubated with FITC-conjugated goat anti-rabbit secondary antibody at 37 °C in the dark for 60 min. Cells were then stained with DAPI for 10 min and mounted with 20 μL of mounting medium for immediate observation under a fluorescence microscope (Yao et al. 2022).

Establishment and treatment of mouse models

A total of 48 male BALB/c nude mice (aged 6–8 weeks, 18–25 g; Vital River Laboratory Animal Technology Co., Ltd., Beijing, China) were used in this study. The mice were housed in separate cages in a specific pathogen-free (SPF) animal laboratory with a 12-h light–dark cycle, 60–65% humidity, and a temperature of 22–25 °C, with free access to food and water. After one week of acclimatization and health observation, the experimental procedures were conducted. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Zhujiang Hospital, Southern Medical University. The animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals and received appropriate housing and management under the supervision of experienced technicians.

Protocol 1: A total of 24 mice were randomly divided into four groups: sh-NC group (xenografted with EC cells with shRNA NC), sh-SIRT1 group (xenografted with EC cells with sh-SIRT1), oe-NC group (xenografted with EC cells with oe-NC), and oe-SIRT1 group (xenografted with EC cells with oe-SIRT1), with six mice per group. Each group received a subcutaneous injection of EC cells (1 × 107 cells suspended in 100 μL PBS) into the back to establish a subcutaneous tumor model. From the 8th day post-injection, tumor width (W) and length (L) were measured every 4 days using a caliper to monitor tumor growth. Tumor volume (V) was calculated using the formula V = (W2 × L)/2. From the 28th day after injection, mice were treated daily with intraperitoneal injections of the synthetic progestin medroxyprogesterone acetate (MA, HY-B0469, MedChemExpress, USA) at a dose of 100 mg/kg. Tumor size was determined every 4 days using a caliper. After 16 days of continuous treatment, mice were euthanized, and tumors were dissected, photographed, and weighed (Gu et al. 2011).

Protocol 2: Each mouse was injected subcutaneously with oe-SIRT1-transduced cells (1 × 107 cells suspended in 100 μL PBS) to establish a subcutaneous tumor model. When the tumor size reached approximately 100 mm3, the mice were randomly divided into four groups: PBS group (control), MA group (100 mg/kg MA), CQ group (25 mg/kg CQ), and CQ + MA group (100 mg/kg MA combined with 25 mg/kg CQ), with six mice per group. Daily intraperitoneal injections were administered, and tumor size was measured every 4 days to calculate tumor volume. After 28 days of continuous treatment, mice were euthanized, and tumors were excised, photographed, and weighed (Chen et al. 2024).

TUNEL assay

Tumor tissue sections from mice were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton-X 100 in PBS for 3 min. The TUNEL staining kit (C1090, Beyotime) was used to stain the cells. Cells were fixed again with 4% paraformaldehyde for 30 min and then incubated with PBS containing 0.3% Triton X-100 at room temperature for 5 min. Next, 50 μL of TUNEL detection solution was added, and the samples were incubated in the dark at 37 °C for 60 min. The samples were counterstained with DAPI (10 μg/mL) for 10 min and then sealed with an anti-fluorescence quenching mounting medium. Cy3 fluorescence (excitation at 550 nm, emission at 570 nm) was observed under a fluorescence microscope (Han et al. 2020). Image Pro Plus 6.0 software was used to calculate the apoptosis ratio.

Immunohistochemical staining

Paraffin-embedded tissue sections were deparaffinized in xylene for 10 min twice and rehydrated in a graded ethanol series (100%, 95%, 70%) for 5–10 min each. Sections were then microwaved in 0.01 M citrate buffer (pH 6.0) for antigen retrieval, followed by cooling to room temperature. After washing with PBS three times for 3 min each, sections were incubated with 3% H2O2 at room temperature to inactivate endogenous peroxidase activity. Subsequently, sections were blocked with normal goat serum (E510009, Sangon Biotech) for 20 min at room temperature.

Sections were incubated with primary antibodies against SIRT1 (ab76039, 1:300), FOXO3 (ab314007, 1:180), LC3B (ab192890, 1:1000), and p62 (ab207305, 1:2000, Abcam) overnight at 4 °C. Sections were incubated with goat anti-mouse or anti-rabbit IgG secondary antibodies for 30 min, followed by incubation with SABC (P0603, Beyotime) at 37 °C for 30 min. DAB substrate was added for color development, followed by counterstaining with hematoxylin. Sections were dehydrated through graded ethanol series and cleared in xylene before being mounted with neutral resin. Observations were made under a brightfield microscope (BX63, Olympus, Japan) (Che et al. 2020).

Tissue immunofluorescence

Tissue sections were fixed with 4% paraformaldehyde for 15–30 min. Subsequently, cells were permeabilized with 0.1% Triton for 15 min and blocked with PBS containing 15% FBS at 5 °C overnight.

FOXO3 nuclear localization staining was further conducted. Sections were incubated with rabbit anti-FOXO3 antibody at 37 °C for 60 min and then incubated with FITC-conjugated goat anti-rabbit secondary antibody at 37 °C for 60 min. Subsequently, tissues were stained with Alexa-488 conjugated goat anti-rabbit antibody and DAPI for 1 h at room temperature. Sections were mounted with 20 μL of mounting medium for observation under a fluorescence microscope (Wagle et al. 2016).

PINK1 and Parkin co-localization staining was adopted to visualize and quantify the localization of proteins and their interactions. Sections were incubated with rabbit anti-PINK1 and anti-Parkin antibodies at 37 °C for 60 min and then incubated with FITC-conjugated goat anti-rabbit secondary antibody at 37 °C for 60 min. Subsequently, sections were stained with DAPI for 10 min and mounted with 20 μL mounting medium for immediate observation under a fluorescence microscope (Yao et al. 2022).

Statistical analysis

Data were derived from at least three independent experiments and are presented as mean ± standard deviation (SD). The independent samples t-test was employed for the comparison between two groups and the one-way analysis of variance (ANOVA) was used for comparisons among three or more groups. If ANOVA indicated significant differences, Tukey's HSD post-hoc test was performed to compare differences between groups. For non-normally distributed data or unequal variances, the Mann–Whitney U test or Kruskal–Wallis H test was applied. All statistical analyses were conducted using GraphPad Prism 9.5.0 (GraphPad Software, Inc.) and R version 4.2.1 (R Foundation for Statistical Computing). A significance level of 0.05 was set for all tests, with two-sided p values < 0.05 considered statistically significant.