Ab109315

The tissue microarray was constructed as described above. The primary anti-CDC6 antibody (diluted 1:1,000; ab109315; Abcam) was used for IHC staining. Two independent pathologists who did not know the clinical pathological data and the clinical results of each patient evaluated the staining intensity of the specimen. The specimens were deparaffinized, hydrated and blocked, and then added into the primary anti-CDC6 goat polyclonal antibody (diluted 1:1,000) and incubated overnight at 4°C. The scores were evaluation positive cells score. The positive cells score: negative: 0–5%; low: 6–25%; medium: 26–50%; high: >50%.

The transfected cells grown on chamber slides were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature or overnight at 4℃, treated with 3% H₂O₂-methanol solution for 10 min, and washed with PBS. After blocking with goat serum for 20 min, cells were incubated with primary antibody against CDC6 (ab109315, abcam, UK) at 37℃ for 2 h, and then incubated with TRITC-conjugated secondary antibody (diluted 1:100) at 37℃ for 1 h. After washing, cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min, and fluorescence was visualized using a confocal laser scanning microscope.

After 72-h transfection, cells were collected, washed twice with pre-cooled PBS, and lysed on ice with buffer containing 10 µl phosphatase inhibitor, 1 µl protease inhibitor, and 5 µl 100 mM PMSE for 30 min. The resulting cell lysate was centrifuged at 14,000 rpm for 15 min at 4℃, and the supernatant was aspirated for protein quantification by the Bradford method. Proteins were separated by SDS-PAGE electrophoresis, electro-transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking at room temperature for 2 h, membrane was incubated with primary antibody overnight at 4℃, and then probed with secondary antibody at room temperature for 1–2 h. The blot was visualized by G: BOX chemiXR5 imaging and analyzed by Gel-Pro32 software. The primary antibodies against CDC6 (ab109315), caspase-3 (ab197202), bcl-2 (ab182858), Bax (ab182734), E-cadherin (ab40772), and ATR (ab13798) were purchased from abcam, UK; anti-INK4 (Cst 74,560) was from Cell Signaling Technology, US.

Proteins were extracted using RIPA lysis buffer containing 1% PMSF and 1% cocktail protease inhibitor. The protein concentration was determined using the BCA assay kit (PC0020, Solarbio, China). The protein samples were separated on a 10% SDS-PAGE gel and then transferred onto a PVDF membrane. The membrane was subsequently incubated with primary antibodies, including FTO (1:2000, ab126605, abcam, UK), POLQ (1:1000, SAB1402530, Merk, Germany), CDC6 (1:2000, ab109315, abcam, UK), CDT1 (1:1000, ab202067, abcam, UK), FEN1 (1:2000, ab109132, abcam, UK), NBN (1:2000, ab32074, abcam, UK), and XRCC1 (1:2000, ab134056, abcam, UK). Subsequently, appropriate secondary antibodies (1:10,000, abcam, UK) were applied for 1 h. The protein bands were visualized using a chemiluminescence imaging system (5200, Tanon, China) for image analysis.

The total proteins in A549 and H1299 cells were extracted by applying the RIPA lysis buffer (Beyotime, Shanghai, China) in the light of the instructions. The concentration of total protein samples was scrutinized by using the BCA kit (Beyotime, Shanghai, China). A weight of 20 μg of the total protein sample were collected and separated with 10% SDS-PAGE. After the blockage by 5% skimmed milk for 1 h at 25° C, the proteins were transferred to the polyvinylidene difluoride (PVDF) membranes. Rabbit anti-primary antibodies were then dropped onto the PVDF membranes to probe the proteins for 12 h at 4° C, including anti-CDC6 (1:1000, ab109315, Abcam, Shanghai, China), anti-CEP55 (1:1000, ab170414, Abcam), anti-TYMS (1:1000, CSB-PA025393GA01HU, CUSABIO, Wuhan, China) and anti-GAPDH (1:1000, CSB-MA000071, CUSABIO). Followed by this, goat anti-rabbit secondary antibody (1:2000, A21020, AmyJet Scientific, Wuhan, China) was utilized for 2 h treatment of the proteins at room temperature. The enhanced chemiluminescent (ECL) kit (AmyJet Scientific, Wuhan, China) was applied for the visualization of the specific protein blots according to the directions. The quantification of proteins was determined by Image Lab software 3.0 (Bio-Rad Laboratories, Hercules, CA, USA).

The media was discarded, and RIPA lysate was added to the cells for total protein extraction. The total protein concentration was measured using the BCA technique (Thermo, Shanghai, China). Protein samples were separated through SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% skim milk at room temperature for 1 h, the membrane was washed three times with TBST and then incubated overnight at 4 °C with the primary antibody at the recommended dilution ratio. Subsequently, the membrane was washed again with TBST and incubated at room temperature for 1 h with the DyLight 800-labeled secondary antibody (ROCKLAND, USA). The membrane underwent three additional TBST washes before imaging using a fluorescence scanning instrument (Odyssey, Danbury, CT, USA). Primary antibodies against FOXM1 (ab207298, dilution: 1/1,000), FAM83A (ab128245, dilution: 1/1,000), CDC20 (ab183479, dilution: 1/2,000), CDC6 (ab109315, dilution: 1/4,000), cyclin B (ab32053, dilution: 1/8,000), H3 (ab1791, dilution: 1/5,000), and GAPDH (ab9485, dilution: 1/2,500) were bought from Abcam for this work.