Proteome profiler human xl oncology array

Eighty-four different cancer-related proteins and the 43 kinases phosphorylation were measured with Proteome Profiler Human XL Oncology Array and Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems). Membranes were developed with Luminata Forte western HRP Substrate (Millipore) and images acquired with a Fujifilm LAS 3000 Imager. Dot intensity was quantified with ImageJ. Values were normalized to internal reference control. A restricted number of candidates were selected by plotting the DLK-A/DLK-C ratio of variation vs. internal reference control.
p53 activity was measured on nuclear extracts with p53 Transcription Factor Assay Kit (Abcam ab207225) following manufacturer’s instruction; Akt phosphorylation levels were measured with AKT1 + AKT2 + AKT3(pT308) ELISA Kit (ab176636) on total cellular extractions; Glucose and Lactate levels, VEGF and MMP9 secretion were measured on supernatant media with Glucose Assay Kit ab102517 (Abcam), L-Lactate Assay Kit (Abcam ab65331), Human VEGF-A ELISA Kit RAB0507 (Sigma) and Human MMP9 ELISA Kit ab100610 (Abcam). Each sample was read in duplicate. For p53, P-Akt, VEGF, MMP9 n = 3 for U3082MG, U3084MG, U3065MG. U3084S, n = 4 (p53, P-Akt), n = 3 (VEGF, MMP9, lactate, glucose).

CWR22RV1 cells were cultured in T75 flasks in complete media containing DMSO (vehicle control) or 2 μM P-SiaFNEtoc for 72 h. Cell lysates were incubated with 600 μL of Lysis Buffer 17 (895943, R&D Systems) at 4 °C for 30 min and concentration was determined with the Pierce™ 660 nm Protein Assay Kit (22662, Thermo Scientific). For conditioned media samples, cells were cultured in serum free media containing DMSO (vehicle control) or 2 μM P-SiaFNEtoc for an additional 48 h then conditioned media was collected in Amicron® Ultra-15 Centrifugal Filter Units (UFC901024, Millipore). The Proteome Profiler Human XL Oncology Array (ARY026, R&D Systems) was performed as per the manufacturer’s instructions, using 200 μg cell lysate or 330 μL conditioned media per sample.

The expression of stem cell- cancer- and stress-related proteins was evaluated with Proteome Profiler Human Pluripotent Stem Cell Array Kit (#ARY010), Proteome Profiler Human XL Oncology Array (#ARY026) and Proteome Profiler Human Cell Stress Array Kit (#ARY018) all from R&D Systems. Protein list is available in Additional file 4: Table S3. Further details are provided in Supplementary Methods, Additional file 1.

Confluent cells were treated with 0.7 μg/mL GolgiStop™ and 1 μg/mL GolgiPlug™ Protein Transport Inhibitors (Thermo Fisher Scientific) for 18 h to prevent cytokine release. Cells were lysed with 1X radioimmunoprecipitation assay (RIPA) buffer with phenylmethylsulfonyl fluoride, sodium orthovanadate and kit inhibitor cocktail (Bio‐Rad Laboratories), prior to protein quantification. Separately, EVs were isolated as described above. Total of 180 μg protein per cell line or EV preparation was incubated with the Proteome Profiler Human XL Oncology Array (R&D Systems) and processed as per the manufacturer's instructions. Membranes were imaged by chemiluminescence using C‐DiGit® Blot Scanner (LI‐COR) and analysed by densitometry using ImageJ software (version 1.50i).

Total protein was isolated from patient-derived cSCC and ANT by adding 500 µL RIPA buffer (50 nM Tris pH 8.0, 150 nM NaCl, 0.5% sodium deoxycholate, 1.0% NP-40, 0.1% SDS with protease inhibitors) per 100 mg tissue and homogenizing (BeadBug, Benchmark Scientific, Seville, Spain) at 20 s intervals for a total of 5 min at 4 °C. Samples were centrifuged at 21,000× g for 20 min, supernatants collected, and re-spun using the same conditions. Protein concentrations were determined using a BCA Protein Assay (Pierce) following the manufacturer’s protocol. A Proteome Profiler Human XL Oncology Array (R&D Systems, Minneapolis, MN, USA) to assess expression levels of 84 human cancer-related proteins was performed on 6 cSCC and 6 ANT samples, in duplicate, using 400 μg of protein following the manufacturer’s protocol. Overall expression of each protein was measured and normalized to vinculin (Supplementary Figure S1).

The expression levels of 84 human cancer-related proteins were evaluated in Weri cells transduced with either GIPR or control lentiviral supernatant using the Proteome Profiler Human XL Oncology Array (R&D Systems, Minneapolis, MN, USA). The expression levels were determined in duplicate, using 200 µg of protein following the manufacturer’s protocol.