Clones of SCC003 expressing either empty vector control, HPV-16 E7, HPV-16 E6, or both HPV-16 E6 and E7, respectively: They were generated by infection of the SCC003 cell line with retroviruses and single cell cloning as follows. Viruses were produced by transfecting Human Embryonic Kidney (HEK) 293T cells with pLXSN (empty vector or containing the HPV-16 E6, E7 or E6&E7 cDNAs, kind gifts from Dr. David Beach) together with pHIT- VSVG and MLV-gag/pol (kind gifts from Dr. Juan Martin-Serrano) using polyethylenimine (Polysciences, Inc.). 72 hours post-transfection, viruses were harvested by removal of the medium and filtration through 0.45um surfactant-free filters (Nalgene). The filtered virus stocks were either frozen at -80C or diluted 1:2 in DMEM/10%FBS containing 8ug/ml Polybrene (to give a final conc. of 4ug/ml) and added to SCC003 cells grown to a confluence of 40-50%. Following overnight incubation, the cells were washed to remove virus and medium was replaced with DMEM/10%FBS. At 48-72hrs post-infection cells were passaged at a ratio of 1:5 into selection medium containing 400ug/ml G418. Following death of all mock-infected cells (approx. 1 – 2 weeks), cells were removed from selection and plated at limiting dilution in 96 well plates to generate single cell clones. HPV-16 E6 and E7 qPCR was conducted as described (Wang-Johanning et al. 2002; Zhao et al. 2005). To assess E6 and E7 expression levels in E6-transduced, E7-transduced and E6&E7-transduced SCC003 cell line clones (and empty vector controls), qPCR was performed on cDNA following reverse transcription (Superscript-II, Invitrogen) of total RNA purified from cells using the miRNeasy kit (QIAGEN) as per manufacturers’ recommendations.
Growth protocol
All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and penicillin/streptomycin.
Extracted molecule
genomic DNA
Extraction protocol
DNA from HNSCC cell linesn were extracted using the QIAamp DNA Blood Mini Kit (QIAGEN)
Label
Biotin
Label protocol
DNA was prepared in a total volume of 20 µl (1 µg). It was then bisulphite converted using the EZ DNA Methylation kit (Zymo Research Corp, Orange, CA 92867, USA, cat No. D5001) before being hybridized to the array.
Hybridization protocol
The Infinium HumanMethylation450 BeadChips were processed by the UCL Genomics core facility according to the manufacturer’s recommendation.
Scan protocol
The Infinium HumanMethylation450 BeadChips were processed by the UCL Genomics core facility according to the manufacturer’s recommendation.
Description
BS-converted DNA
Data processing
The scanned data and image output files were managed with the Genomestudio software (version 1.9.0) (Illumina®, Inc., San Diego, CA 92121, USA).
