Proteome profiler array kit

For biochemical analyses, exponentially growing cells were seeded in complete medium and treated the day after with the drug at the indicated concentrations. After 48h, cells were processed for RTK analysis using the Proteome Profiler Array Kit (ARY001/ARY001B, R&D systems) according to the manufacturer's instructions, or for total protein extraction, or immunoprecipitation as previously described in details [68 (link), 69 (link)]. Otherwise, proteomic analysis was performed on lysates from serum starved cells, or on lysates from frozen tumors analogously processed after pulverization by the Mikro-Dismembrator II (B. Brown Biotech International, Melsungen, Germany). For validation experiments, immunoprecipitates or cell lysates were prepared, separated by SDS-PAGE, transferred on nitrocellulose and analyzed by western blotting as described [68 (link)], using the indicated antibodies.

2-Hydroxymelatonin (2(OH)Mel), 6-hydroxymelatonin (6(OH)Mel), bovine serum albumin (BSA), ethanol (EtOH), glucose, melatonin, and sodium pyruvate were purchased from Sigma (St. Louis, MO, USA). AFMK and GlutaMAX™ Supplements were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Other reagents were supplied as follows: EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (Millipore Merck KGaA, Darmstadt, Germany); RNA Miniprep Kit (Agilent Technologies, Santa Clara, CA, USA), high capacity cDNA Reverse Transcription Kit (Applied Biosystems), DyNamo Flash SYBR Green qPCR Kit (Thermo Scientific, Waltham, MA, USA), KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA), the Cell Mito Stress Test media was supplemented with 2 mM GlutaMAX™ Supplement (L-alanyl-L-glutamine dipeptide in NaCl), Proteome Profiler Array kit (R&D Systems, Minneapolis, MN, USA), lactate colorimetric assay kit (Cell Biolabs, Inc. San Diego, CA, USA), TeloTAGGG Telomerase Elisa assay (Roche, Basel, Switzerland), and a set of human primers for real-time PCR (Eurofins Genomics, Ebersberg, Germany).

The phosphorylation level of kinases was determined with the Proteome Profiler Array Kit (R&D Systems) according to the manufacturer’s instructions. Protein concentrations were determined by the Bradford protein assay. To block non-specific sites, each membrane was incubated in an array blocking buffer for 1 h. 3, 3′- (3, 5-DCPBC), and DMSO treated cell lysates (334 µL cell lysate/1 mL of array buffer corresponding to 200 µg protein lysate) were applied on membranes and incubated overnight. Thereafter, membranes were washed with 1X washing buffer followed by incubation with 20 mL of the detection antibody for 2 h on a shaker at room temperature. Membranes were thoroughly rinsed with washing buffer thrice and further incubated with Streptavidin-HRP for 30 min at room temperature. Membranes were washed with 1× washing buffer for 10 min, and after that, all membranes were simultaneously exposed to SignalFire plus chemiluminescent reagents for 1 min. Phospho-kinase array data were developed on Vilber FusionFx Chemiluminescence Imager for 1 to 10 min with multiple exposure times.

Samples were sorted (purity) into EpCAM positive populations using the BD FACS Aria II, the cells lysed, and the proteome profile determined using the Proteome Profiler Array Kit (R&D Systems) as per the manufacturer’s instructions. Array membranes were analysed and quantified using the Syngene Chemigenius II system (Cambridge, UK)

To evaluate the expression of phosphorylated RTKs, a Proteome Profiler Array Kit (R&D Systems) comprising spotted antibodies for 49 kinase phosphorylation sites was used to perform the phospho-RTK array according to the manufacturer’s protocol.