genotype: Grhl3 conditional knockout process center: Host and Tumour Profiling Unit (HTPU, Cancer Research UK, Edinburgh, UK)
Treatment protocol
Lentiviral expression constructs were mixed with the packaging plasmids pMDLgRRE, pRSV-Rev, pCMV-VSVG (Addgene) and transfected into lenti-X293T cells (Takara Bio, #632180) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Virus-containing conditioned media was collected 48–72 hours after transfection and filtered using a 0.45-µm filter. Viral conditioned media was supplemented with polybrene and applied to sub-confluent cells. Conditioned media was replaced with fresh cell culture media 6 hours post transduction. Double-positive GFP and mCherry-expressing cells were sorted using a FACS Calibur cell sorter 72 hours post transduction and then expanded in culture. Doxycycline (Dox) Hyclate (Sigma Aldrich) was added (1µg/ml) to the culture media to induce CRISPR-Cas9-mediated gene deletion for downstream analysis.
Growth protocol
The normal oral epithelial cell line OKF6 was purchased from the Harvard Skin Disease Research Centre (HSDRC, Boston MA). The oral cancer cell lines SCC15 (CRL-1623) and SCC25 (CRL-1628) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). All cell lines were authenticated and validated by short tandem repeat (STR) profiling and tested negative for mycoplasma contamination (33). OKF6 and SCC25 were cultured in keratinocyte serum-free medium (K-SFM, GibcoTM) supplemented with growth factors (25 mg/mL BPE, 0.2 ng/mL EGF and 0.3 mM CaCl2) and 1% penicillin-streptomycin (P/S). SCC9, SCC15, and CAL27 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GibcoTM) with 10% foetal bovine serum (FBS) and 1% P/S. All cell lines were cultured at 37°C in a 5% CO2 humidified incubator and maintained at less than 25 passages.
Extracted molecule
protein
Extraction protocol
Protein lysates from human HNC cells were extracted using the RPPA lysis buffer and quantified by the Pierce Coomassie Plus (Bradford) Assay Kit (ThermoFisher Scientific, Cat No. 23236).
Label
biotin
Label protocol
Sample lysates were run at the Host and Tumour Profiling Unit (HTPU, Cancer Research UK, Edinburgh, UK).
Hybridization protocol
Sample lysates were run at the Host and Tumour Profiling Unit (HTPU, Cancer Research UK, Edinburgh, UK).
Scan protocol
Sample lysates were run at the Host and Tumour Profiling Unit (HTPU, Cancer Research UK, Edinburgh, UK).
Description
SAMPLE 21
Data processing
RPPA Relative Fluorescence Intensity (RFI) values were calculated by the Zeptoview software. A weighted linear regression through the dilution series was used to calculate the sample fluorescence intensity value which was then normalised to the reference BSA grid to account for intra-array spatial variation. Each intensity value was corrected to the background signal and the secondary antibody controls to validate the RFI value for each sample/antibody combination. ‘0’ accounted for RFI values where the primary antibody signal was lower than the signal emitted by the secondary antibody alone. 1e-9 was added to all RFI values for statistical analysis. Please note that PMR-125_NormToSec.RawData.xlsx is a direct output from ZeptoVision, which automatically normalises to the background.
