The functional role of PRRT2 was assessed in RWPE-1, HCT116 and PNT2C2 cell lines expressing the truncated form of PRRT2 (ΔPRRT2), the wildtype PRRT2 (wtPRRT2) and the control GFP. These cell lines were propagated in medium supplemented with 1 μg/mL puromycin (Millipore). Briefly, cells were plated in 96-well culture plates at a density of 1000 cells per well (100 μl). Proliferation was assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), according to manufacturer's protocol. Cell cycle analysis was performed by propidium iodide staining of the cell lines involved. Following cell pellet collection, fixation with 70% ethanol was performed. Fixated cells were washed in PBS-0.05% Tween20 and ressuspended in 500 μL of PBS-0.05% Tween20, RNases and PI. Samples were measured in FACS calibre following a 15 minute incubation with PI. Apoptosis was assessed using the ApoLive-Glo™ Multiplex Assay (Promega), which measures both the number of viable cells as a marker of cytotoxicity and caspase activation as a marker of apoptosis within a single assay well. The assay was performed according to the manufacturer's protocol. As positive control of apoptosis an apoptosis inducer set (Millipore) was used. A mix containing 700x dilutions of Actinomycin D (10 mM), Camptothecin (2 mM), Cycloheximide (100 mM), Dexamethasone (10 mM) and Etoposide (10 mM) was supplemented for 24h to induce apoptosis. The QCM Chemotaxis Cell Migration Assay, 96-well (8 μm), fluorimetric (Millipore) was performed according to the manufacturer's protocol. CytoSelect™ 24-Well Cell Invasion Assay, Collagen I was used to determine cell invasion according to the manufacturer's protocol. Full paper
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A mononucleotide repeat in PRRT2 is an important, frequent target of mismatch repair deficiency in cancer
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