Supplementary information
Materials and Methods
The information for two liver cancer cell lines, BEL7402 and QGY 7703
These two HCC cell lines have been established in China before 33 years ago. Their biological characteristics were stated as follows: BEL7402 cell line was derived from hepatic carcinoma specimen of an operated male patient.It was rapidly adherent growth, and the doubling time was 20-26 hours. It appeared as epithelial-like cell in morphology, in addition to desmosomes, the presence of cytoplasmic to nofibrills which was typical of epithelial cell by electron microscopy. The ultrastructural feature was different from normal human liver cell, but similar to clinical hepatoma cell. They had hypotriploid chromosone number with one abnormally long acrocentric chromosome present. AFP was detected intracellularly by the indirect immunofluorescent method.BEL7402 cell line is a not well differentiated epithelial-like malignant cell and growing rapidly.
In 1981, QGY 7703 cell line was constructed from the liver cancer tissue of a 35 years oldwoman.It was an epithelial-like celland rapidly adherent growth,doubling time was 20.5 hours. Chromosome number changed greatly and much heteroploid.Polyploid cells had good capacity of xenografts.AFP was positive via indirect immunofluorescence test. Submicroscopic structure aspect, nuclear and cytoplasmic ratio is high,including big polymorphic nucleus.
Patients and Tissue Specimens
The fresh tissues for protein extraction were frozen in liquid nitrogen immediately after resection, while those for RNA extraction were immersed in RNA later (Ambion,Inc,USA) and stored at 4°C overnight then frozen in liquid nitrogen.
RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
Briefly, total RNA was extractedusing the TRIZOL reagent(Invitrogen)according to themanufacturer’s protocol. First-strand cDNA was synthesized using PrimeScript® RT reagent Kit with gDNA Eraser(Takara) and qPCR was conducted for detection of CBX4 mRNA which was amplified using aSYBR Green PCR Kit (Takara)on Bio-Rad Laboratories and a Bio-RadCFX96 sequence detection system.Primers for qRT-PCR are following,
Primers for CBX4
forward,5’ GCAGAGTGGAGTATCTGGTGA 3’
reverse,5’AGCTTGGCACGGTTGTCAG 3’
Primers forGAPDH
forward, 5’ACAGTCAGCCGCATCTTCTT 3’
reverse, 5’GACAAGCTTCCCGTTCTCAG3’
Primers for p16,
Forward: 5’GAAGGTCCCTCAGACATCCCC 3’
Reverse: 5’CCCTGTAGGACCTTCGGTGAC 3’
Primers for PCNA,
Forward: 5’ ACACTAAGGGCCGAAGATAACG 3’
Reverse: 5’ ACAGCATCTCCAATATGGCTGA3’
Primers for cyclinE2,
Forward: 5’TCAAGACGAAGTAGCCGTTTAC3’
Reverse: 5’TGACATCCTGGGTAGTTTTCCTC3’.
siRNA Transfection
siRNAs were purchased from Guangzhou RiboBio Co.,Three targeting and siRNA sequences for CBX4 as follows:
#1 5’ AGATGAAGATAGTCAAGAA 3’
#1siRNA (homo)
5’ AGAUGAAGAUAGUCAAGAAdTdT3’
5’ UUCUUGACUAUCUUCAUCUdTdT 3’
#2 5’ TGAAGATAGTCAAGAACAA 3’
#2siRNA (homo)
5’ UGAAGAUAGUCAAGAACAAdTdT 3’
5’ UUGUUCUUGACUAUCUUCAdTdT 3’
Western blotting antibodies
Rabbit anti-human CBX4 polyclonal antibody(1:1000;Sigma-Aldrich,HPA008228)，Rabbit anti-human PCNApolyclonal antibody(1:5000;Bethyl Laboratories, Inc,A300-276B), Rabbit anti-human p16 polyclonal antibody(1:500;BD Biosciences,#554079), Rabbit anti-human CyclinE2 polyclonal antibody(1:1000;Cell signailingTec,#4132S) ,Mouseanti-humanβ-actin monoclonal antibody(1:1000; Bioworld Technology, Inc, BS6007M)
Immunohistochemistry(IHC) protocol
Briefly, paraffin-embedded specimens were cut into 4 μm sections and baked at 60℃ for 2 hours.The slides were deparaffinized with xylenes and rehydrated in ladder ethanol, then were submerged in EDTA buffer(pH 8.0) and high pressure boiling for 4minutes for heat mediated antigen retrieval.The endogenous peroxidase was quenched by 3% hydrogen peroxide for 15 minutes. Goat nonimmunalserum was applied to block non-specific binding. The sectionswas incubated with antibody against CBX4 (1:75 dilution,Sigma-Aldrich) at 4°C overnight. Antigen was detected using secondary anti-rabbit polymer HRP and DAB chromagen kit(Genetech) and followed by counterstained with hematoxylin.
Cell collection and FACS Analysis
Briefly,cells were harvested by trypsinization andcollected by centrifugation. Cells were washed once withphosphate-buffered saline (PBS) and fixed in 1ml of 70%ethanol at 4℃. Cells were washed once with PBS/1%bovine serum albumin (BSA) followed by incubation with 1ml ofPBS/1% BSA containing 30 mg/ml propidium iodide and0.25 mg/ml RNase A for 30 min at room temperature. Cellswere analyzed for DNA content by FACS using acytomics FC 500 (Beckman Coulter, Fullerton, CA, USA).The data were analyzed using Multicycle AV for windowssoftware (Beckman).
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