References to supplementary tables and figures are indicated with prefix ‘S’. A comprehensive list of antibodies used in this study is presented in Table S1.
Cell culture and stable cell line generation
Human ovarian cancer cell lines A2780, C13, ES-2, HO8910, OVCAR-3, and SKOV3 were purchased the American Type Culture Collection (ATCC, Manassas, VA, USA). Human normal ovarian epithelial cell line IOSE was supplied by Heng-Yu Fan, Zhejiang University [37]. Cells were grown in DMEM (Gibco | Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco | Thermo Fisher Scientific) and 1% penicillin-streptomycin (Gibco | Thermo Fisher Scientific) at 37 ℃ in humidified atmosphere composed of 5% CO2 and 95% air (standard culture conditions).
DCAF13-deficient cells were established using CRISPR/Cas9 technology. The guide RNA sequences used for targeting human DCAF13 were: human DCAF13 - 1: 5’- AGCGGGACAGCAGTGAGCCC-3’; human DCAF13 - 2: 5’-GATGTGGATTACTCTCCCAC-3’. The construction of DCAF13-deficient cell lines was previously described [11].
Cell proliferation and colony formation assays
A total of 1×105 cells were seeded per well in a 6-wells plate (Corning, NY, USA) (n = 3 per group). The cells were counted by hemocytometer at 24, 48, and 72 hours after seeding. Cell count was plotted as a function of time after seeding.
The colony formation assay was conduct on soft agar. Six-wells plates were coated with 1.5 mL of 0.5% agar (Sigma-Aldrich, St. Louis, MO, USA) base layer. Subsequently, a suspension of 2 ×103 cells in 1.5 mL of 0.35% top agar was carefully added. To provide nutrients to the cells, 2 mL of cell culture medium was transferred onto the top layer twice a week. After a period of 3 weeks following plating, colonies were stained with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO, USA) dissolved in PBS and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Scratch and Transwell assays
A total of 5×105 cells were seeded in 6-wells plates in medium supplemented with 10% FBS. Once cells reached 90% confluence, the monolayer was scraped with a with 10-µL pipette tip across the center of each well and the cells were washed once with PBS. Next, 2 mL of fresh serum-free medium was added to each well to starve the cells. The plates were imaged immediately after scratching and washing (baseline) and at 24 hours using an inverted phase contrast microscope (CKX53, Olympus, Tokyo, Japan). The extent of cell migration was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and data are presented as percentage of cell-occupied area in the scratch channel at 24 hours versus baseline.
Migration experiments were conducted in 24-wells plates using Transwell chambers equipped with 8-μm pore filters (Corning, NY, USA). Cells (1×104) were resuspended in 300 μL of FBS-free medium and transferred into the upper chamber. Next, 500 μL of medium containing 10% FBS was added to the lower chamber. After 24 hours, cells were fixed in methanol for 5 minutes. Stationary cells in the upper chamber were removed with cotton swabs. The cells that had migrated were stained with hematoxylin and quantified using ImageJ software (National Institutes of Health).
Western blotting
Protein from cells and tissues was extracted using RIPA lysis buffer (Beyotime Biotechnology, Haimen, China) and quantified with a bicinchoninic acid assay (BCA assay kit; Beyotime Biotechnology). 20 μg protein per well was separated using SDS-PAGE, transferred to PVDF membranes (Merck | Millipore, Burlington, MA, USA), and blocked using 5% powdered milk for 1 hour at RT. PVDF membranes were incubated overnight at 4 °C with primary antibodies against proteins of interest. Subsequently, the samples were incubated with a secondary antibody, specifically an anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA). The resulting bands were then visualized using an enhanced chemiluminescence detection kit (Merck | Millipore). Data acquisition was carried out using an Imager 680 (Amersham | GE Healthcare, Chicago, IL, USA). Image analysis and quantification using ImageJ software (National Institutes of Health).
Co-immunoprecipitation assay
Cells were lysed in cell lysis buffer for Western and IP (Beyotime Biotechnology). Cell lysates were spiked with 50 μL of Protein A/G magnetic beads (MCE Magnetic, Mianyang, China) and washed 3 × with 400 μL binding/washing buffer (1 × PBS + 0.5% Tween-20). The corresponding primary antibody was incubated with magnetic beads at 4 ℃ for 4 hours, and subsequently the cell lysates were transferred into antibody-magnetic bead complex solution and incubated overnight at 4 ℃. The beads were thoroughly washed 5 × with a binding/washing buffer, followed by suspension in 1 × loading buffer, and heated at 95°C for 5 minutes. The resulting samples were then analyzed by Western blot.
Protein Identification Analysis
This experiment was performed using 293T cells. DCAF13 was overexpressed by transfecting the DCAF13-FLAG plasmid into 293T cells. When the cells grew to 80%, the cells were collected and lysed in cell lysis buffer for Western and IP (Beyotime Biotechnology). The cell lysates were supplemented with 50 μL of Protein A/G magnetic beads (MCE Magnetic) and subjected to three washes using 400 μL of binding/washing buffer (1 × PBS + 0.5% Tween-20). Sequently, the corresponding primary antibody was incubated with the magnetic beads 4 hours. Following this, the cell lysates were transferred into a solution containing the antibody-magnetic bead complex and incubated overnight at 4 ℃. The beads were thoroughly washed five times with a binding/washing buffer, followed by suspension in 1 × loading buffer, and heated at 95°C for 5 minutes. Control experiments using IgG antibodies. Proteins were separated using SDS-PAGE. The gel was stained with Coomassie Brilliant Blue for 30 minutes and destained by deionized water to remove background. The obtained samples were subjected to protein identification using LC-MS/MS by APT Biotechnology (Shanghai, China).
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cultured cells or murine tumor tissues using TRIzol reagent (Invitrogen | Thermo Fisher Scientific) (n = 3 per group). Extracted RNA was converted to cDNA through reverse transcription using Prime Script RT Reagent Kit (Takara Bio, Shiga, Japan). Real-time PCR analysis was performed with TB Green Master Mix Kit (Takara Bio) on a Realplex2 PCR System (Eppendorf, Hamburg, Germany). The mRNA levels of each gene were standardized to the expression levels of the housekeeping gene β-actin. Primer information is presented in Table S2.
Plasmids and RNA interference
Expression constructs coding for mouse Dcaf13 cDNA (Flag-DCAF13), Flag-DCAF13 SOF∆, Flag-DCAF13 WD∆, Flag-DDB1, and Myc-Ub plasmids were kindly provided by Dr. Heng-Yu Fan [38]. This protein is characterized by the presence of seven WD40 repeats at its N terminus and a SOF1 domain located at the C terminus. Human FRAS1 cDNA was cloned by Miaoling Biotechnology (Wuhan, China). FRAS1 is a remarkably conserved protein consisting of 1976 amino acid residues, with a molecular weight of 217 kDa.
A total of 2×105 cells were seeded in six-well plates for 24h. Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection. After 48 hours of transfection (final siRNA concentration 80 nM), the cells were collected and analyzed by qRT-PCR or Western blot to assess interference efficiency. The siRNA sequences are listed in Table S3.
Flow cytometry analysis
Cells (1×106) were fixed with 70% ethanol for 24 hours. After centrifugation and a dual washing step with PBS, the cells were resuspended in 500 μL of PI + RNase staining buffer (BD Biosciences, Franklin Lakes, NJ, USA) and incubated at 37 ℃ in the dark for 30 minutes. Cells were assayed by flow cytometry (model flow cytometer; BD Biosciences). Data were analyzed using ModFit software (Verity Software House, Topsham, ME, USA).
Mouse xenograft models
All animal experiments were approved by the Jiaxing University’s institutional review board (registration no. JUMC2020-069). Specific pathogen free female BALB/c nude mice, aged 6-8 weeks, were obtained from Jiangsu Jicui Yaokang Biotechnology Co. (Nanjing, China). Animals were housed in a room with 12-hour light/dark cycles in individually ventilated cages with ad libitum access to sterilized food and water. The animals were treated in accordance with institutional guidelines and the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (8th edition).
The mice were randomly assigned to three groups and anesthetized with ether. Subcutaneously, One group was injected with wild-type ovarian cancer cells in the right dorsal flank (n = 6/group), and the other two groups was injected with DCAF13-deleted cells (single bolus of 5 × 106 cells in PBS) in the right dorsal flank (n = 6/group). The tumor size of the mice was measured with a caliper every 2 - 3 days. The tumor volume was calculated using the formula: (width)2 × height × 0.523 [39]. Nude mice were killed by cervical dislocation. A tumor diameter of > 15 mm constituted a human endpoint. Resected tumor tissue was fixed in 4% paraformaldehyde or stored at -80 ℃ until further use.
Histochemistry and immunohistochemistry
Paraffin-embedded human tissue samples from ovarian cancer tissue and paracancerous tissues were provided by the Affiliated Hospital of Jiaxing University (approval no. LS2020-148). Human ovarian cancer tissue microarrays were purchased from Shanghai Outdo Biotech Co. (Shanghai, China). Clinical information was provided by Shanghai Outdo Biotech Co.
Method for analyzing experimental data generated by immunohistochemical experiments on tissue chips: 1. Staining intensity score: 0 points (negative), 1 points (1+), 2 points (2+), 3 points (3+). 2. Staining positive rate: 0 points (negative), 1 point (1-25%), 2 points (26%-50%), 3 points (51-75%), 4 points (76%-100%). 3. Grouping of high-low expression analysis: The product of "staining intensity score" and "staining positive rate score" was used as the total score for grouping, <6 was divided into low antibody expression group, >=6 was divided into high antibody expression group.
Fixed mouse-derived tumor tissue was thawed, embedded in paraffin, sliced into 5-μm thick sections (Leica, Wetzlar, Germany), deparaffinized, and stained with hematoxylin and eosin (H&E). For immunohistochemistry (IHC), the deparaffinized sections underwent 10-minute incubation in 0.3% H2O2. Antigen retrieval was performed using 10 mM sodium citrate (pH = 6.0) for 15 minutes. Subsequently, the sections were incubated overnight at 4 °C with primary antibodies against Ki67 (1:400), p-histone H3 (1:200), and cleaved caspase-3 (1:200) (Table S1). Next, a biotinylated and peroxidase-conjugated secondary antibody was applied for 30 minutes (1:400, Cell Signaling Technology). The sections were counterstained utilizing a Vectastain ABC kit and a 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA).
Immunofluorescence staining
Cells (5×104) were seeded in 24-well plates, fixed with 4% paraformaldehyde for 30 minutes, and blocked using 5% bovine serum albumin for 1 hour. Subsequently, the cells were incubated with primary antibodies against Ki67 (1:400), p21 (1:800), and p-H2AX (1:400) (Table S1). Primary antibodies were removed with a single washing step, and cells were incubated with secondary antibodies labeled with Alexa488 or Alexa594 (Abcam, Cambridge, UK). Next, cells were counterstained with DAPI (Beijing Solarbio Science & Technology Co., Beijing, China). Digital images were captured with a confocal laser scanning microscope (FV3000, Olympus).
Statistical analysis
GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Mean ± standard deviation represented the data and their differences. Samples with n < 8 were subjected to analysis using nonparametric tests. The normal distribution of data was assessed using ANOVA. The correlation between DCAF13 expression and clinicopathological characteristics was assessed using both the Chi-square test and Fisher's exact tests. Using the Kaplan-Meier method, survival rates after tumor removal were calculated, and differences in survival curves were evaluated using the Log-rank test. Additionally, a multivariate survival analysis using the Cox proportional hazard regression model was performed, integrating all relevant traits found in the univariate survival study. A P-value of < 0.05 was considered statistically significant.