Cell culture and transfections
Human CC cell lines C33A, CASKI, SiHa, and HeLa, as well as the human cervical epithelial immortalized cells (H8), were acquired from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and cultured in Dulbecco-modified Eagle medium (Invitrogen) augmented with penicillin 100 (U/mL)/Streptomycin (100 g/mL) and 10% fetal bovine serum (Gibco, USA) at 37℃ and 5% CO2.
Immunohistochemistry
The detailed protocol for immunohistochemistry has been described in our previous research(26). Briefly, the samples in glass slides were dehydrated, deparaffinized in xylene, heated in a microwave with citrate buffer (pH 6.0) for 15 min, blocked with 10% normal goat serum for 30 min, and then treated with 3% H2O2 to inhibit endogenous peroxidase activity for 10 min. The slides were then incubated overnight at 4°C with an anti-CDCA8 antibody (1:100, Proteintech Group, Inc., Wuhan, China), stained with diaminobenzidine (DAB; Sigma-Aldrich), counterstained with hematoxylin, washed with PBS, mounted using permount, dehydrated, and cleared in xylene.
Data collection and processing
The normalized gene expression data on CESC (Cervical Squamous Cell Carcinoma and Cervical Adenocarcinoma) were acquired from the TCGA (The Cancer Genome Atlas) (http://gdac.broadinstitute.org/) database. The differential expression of CDCA8 was assessed based on limma |(log2 (fold change)| > 0.58 and BH-adjusted p-value < 0.05.
RNA extraction and quantitative reverse transcription-PCR (qRT-PCR)
Total cellular RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA), which was then utilized for generating cDNA using SuperScript III (ThermoFisher). The mRNA expression of CDCA8 and GAPDH was detected by qRT PCR using SYBR Green qRT PCR kit (Roche, Basel, Switzerland), Applied Biosystems Real-Time PCR system (Life Technologies, Carlsbad, CA), and QuantStudio 12 K Flex software. GAPDH was employed as a reference point. The 2−ΔΔCt method was utilized to calculate the relative gene expression levels. Each experiment is repeated thrice. The sequences of primers utilized were:
GAPDH: TGACTTCAACAGCGACACCCACACCCTGTTGCTGTAGCCAAA121bp.
CDCA8:TTGAGTCAGACAGGCAGAACCTTCCTCCAAGGGCGAAGTAG 120bp.
MTT assay
The transiently transfected cells (3,000/well) were seeded in 96-well plates and cultured for 1, 2, 3, 4, or 5 days. Then, 20 µL of MTT working solution in the medium was added to each well for 4 hours, followed by the removal of media and the addition of dimethyl sulfoxide (200 µL) to dissolve the formazan crystals. The absorbance was detected at 570 nm (A570) using a microplate reader (BioRad, Hercules, USA).
Colony formation assays
Cells (1000/well) were seeded in 6-well plates, with 3 wells per group. After 14 days of cultivation at 37℃, colonies were stained with crystal violet solution and washed thrice with PBS. Then, colonies with > 50 cells in each well were counted under the microscope.
Wound-healing assays
Transfected cells were cultured in 6-well plates in a complete medium until 90 - 100% confluency. Then, a P200 pipette tip was used to wound the cell monolayers. The cells were then washed to remove debris with PBS and imaged from the same wounded area at 0, 24, and 48 hours.
Transwell migration assays
Cells (4 × 105/well) were seeded in the top chamber of a 24-well Transwell plate (Corning Incorporated, USA) in 1% FBS-containing medium for transfection. In the bottom chamber, a complete medium was added. After incubating for 24 hours in normal conditions, cells on the upper part of the filter were swabbed using a cotton swab, while the cells on the lower surface of the filter were fixed with 4% paraformaldehyde for 20 min, stained with 1% crystal violet for 30 min, and then visualized under the light microscope. The average number of migrating cells was calculated from 5 different fields of view.
Western blot analysis
Briefly, the cells were lysed using RIPA buffer (150 mM NaCl, 25 mM Tris HCl, pH 7.4, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 2mm EDTA) augmented with protease and phosphatase inhibitor cocktail (Roche, France). The acquired proteins (50 µg) were then subjected to SDS-PAGE separation and treated with specific antibodies for immunoblotting (Supplementary Table 1).
Co-immunoprecipitation (co-IP)
Briefly, the IP lysis buffer containing protease inhibitor cocktail was used to lyse SiHa cells NC or Flag CDK1 overexpression cells. The protein lysates were then incubated overnight at 4°C with CDCA8 or Flag primary antibody and then with protein A/G agarose beads for 4 h at 4°C. In addition, 5% cell extract was saved as the input. After overnight incubation, the immunocomplexes were washed thrice with PBST (pH 7.4 PBS with 0.1% Tirton-X100) and then resolved by SDS-PAGE. The bound proteins (CDK1 or CDCA8) were eluted by boiling with 2 × SDS loading buffer.
Tumorigenicity assays in nude mice
The Shanghai Lab Animal Research Center (Shanghai, China) provided the naive mice (age = 4 - 6 weeks), which were housed in the Experiment Animal Center of the Central South University (Changsha, China) at a 12:12-hour light-dark cycle in a temperature-controlled room and ad libitum access to food and water. Stably transfected cells (2 × 106) were added to 0.2 mL RPMI-1640 culture medium and subcutaneously injected into the upper right back of mice for tumorigenicity testing. Each mouse's tumor volume was measured every 3 days using formula 3.14/6 × L × W × W. All mice were sacrificed by cervical dislocation at the end of the experiment at 3 weeks after the injections, and the tumor size was measured with a caliper. Surviving animals after the experiment were handed over to qualified institutions for harmless treatment. The Animal Care and Use Committee of Xiangya Second Hospital of Central South University (Changsha, Hunan, China) (approval number: 20240576) approved all animal operations which strictly followed institutional policies and approved experimental operation guidelines.
Statistical analyses
SPSS 16.0 software was used for all of the statistical analyses. To determine the significance differences between the experimental and control groups, two-sided unpaired Student's t-tests were used. A p-value of 0.05 was deemed statistically significant.