Cell culture
The A431 cell line was obtained from the Institute of Cell Biology of Shanghai, Chinese Academy of Sciences (Shanghai, China). The HSC-1 cell line was obtained from Beijing Zhongyuan Ltd. (Beijing, China). The cells were cultured in DMEM (Gibco, 10566016) supplemented with 10% FBS (Gibco, 10091148) and incubated at 37°C in a humidified 5% CO2 atmosphere.
Celigo cell counting assay
A431 and HSC-1 cells were seeded into 96-well plates at 3×103 cells/100 µL/well. The cells were continuously monitored for 5 days by a Celigo Imaging Cytometer (Nexcelom). The number of GFP-positive cells in each well was accurately calculated.
Woundhealing assay
A431 and HSC-1 cells were seeded in a 24-well plate. When the cells reached 90% confluence, they formed a confluent monolayer. A wound was formed by dragging the tip of the pipette. Cell migration images were acquired at 0 and 36 h by microscopy. Migration area = 24 h-cell pixel area − 0 h-cell pixel area.
Transwell assay
The transwell upper chambers (8-µm pores, BD Biosciences) were coated with Matrigel. A431 and HSC-1 cells were seeded into the transwell upper chamber at a density of 1×104 cells/well in 100 µL of 5% FBS culture medium, and the lower compartment contained 400 µL of culture medium supplemented with 20% FBS. The cells were incubated at 37°C for 24 h. The upper chamber was washed with PBS, and the cells were removed. The migrating cells were fixed with methanol for 30 min and stained with 0.1% crystal violet for 20 min. Finally, the cells were observed with a microscope, images were captured, and the migrating cells were counted.
Flow cytometry apoptosis assay
A431 and HSC-1 cells were infected with lentivirus for 48 h. The cells were harvested and rinsed three times with PBS. Then, the cells were resuspended in 1× binding buffer and incubated with FITC-annexin V for 25 min and PI for 5 min in the dark at room temperature. The cells were detected by flow cytometry. The cell apoptosis rate was analyzed with FlowJo software.
Q-PCR assay
Total RNA was extracted with TRIzol solution (Invitrogen, 15596026). Reverse transcription of cDNA was performed with a M-MLV Reverse Transcriptase (Promega, M1705). qPCR was performed in triplicate with AceQ qPCR SYBR Green Mastermix (Vazyme, Q111-02). The following qPCR primers were used:
CENPO: 5′- TGCTTTTGAGGGGAACCTATTG-3′ (Forward) and 5′- GGGGAATG AAGACTGGGACT-3′ (Reverse); GAPDH: 5’- TGACTTCAACAGCGACACCCA-3’ (Forward), 5’- CACCCTGTTGCTGTAGCCAAA-3’ (Reverse).
Tumor formation assay in nude mice
Female SCID mice (4–6 weeks old) (Cavens, China) were raised in a pathogen-free facility. A431 cells were harvested, washed, and resuspended in PBS. The cells (5×106 cells/100 µL) were injected into the left armpit of each mouse. There were 5 mice in each group. The tumor volume was measured with a Vernier caliper every three days.
The mice were sacrificed and dissected, the tumors were photographed and weighed, and the tumor volumes were calculated using the following equation: length×width2×0.5. All animal experiments were carried out with the approval of the Animal Care and Use Committee of The Air Force Medical Center, PLA.
IHC assay
Tumor tissue was fixed, embedded, and sliced. The expression level of CENPO was detected by IHC with an anti-CENPO antibody (1:500, Proteintech, 20611-1-AP). Images were acquired with a microscope (Nikon, Tokyo, Japan).
Statistical analysis
The experimental data were collected and are expressed as the means ± standard deviations (SD), and the data were analyzed using GraphPad Prism 8.0 software. Significant differences between two groups were analyzed using Student’s t test. Each experiment in this study was repeated at least 3 times. *p value < 0.05, ** p value < 0.01 and *** p value < 0.001.