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Summary FLT3 receptor‐associated signalling plays a role in proliferation and leukaemia. The transcription factor C/EBPβ may be involved in malignancy with its alternative translation product C/EBPβ‐LIP. We investigated a potential connection between FLT3 signalling and the C/EBPβ system in FLT3‐internal tandem duplication (ITD)‐positive leukaemia cells and FLT3‐ITD‐ or FLT3‐wild type (WT)‐transfected 32D cells. In FLT3‐ITD‐positive cells or when ITD sequences were inserted into the FLT3‐WT receptor, significant LIP levels, increased LIP/LAP ratios, and enhanced proliferation rates were detected, which were reduced by FLT3 inhibition. In FLT3‐WT cells, incubation with FLT3 receptor ligand (FL) also elevated LIP, LIP/LAP, and proliferation, albeit to a lesser extent. CEBPB‐directed siRNA decreased both LIP and proliferation rates in FLT3‐ITD‐positive and FL‐stimulated FLT3‐WT‐positive cells. PI3K inhibition affected ITD‐associated and FL‐induced LIP levels. Rapamycin, an inhibitor of mTOR involved in CEBPB translation, completely blocked the increase in LIP in FL‐stimulated FLT3‐WT‐ but not FLT3‐ITD‐positive cells. In contrast, the ITD‐associated LIP elevation was mediated by p90‐ribosomal‐S6‐kinase. This is the first report showing a LIP increase in the presence of ITD or following FL exposure. Our data suggest fundamental differences in the signalling cascades activated via ITD mutations or following FL stimulation, indicating the need for adapted molecular therapy.

Peptide and protein micropatterns are powerful tools for the investigation of various cellular processes, including protein-protein interactions (PPIs). Within recent years, various approaches for the production of functional surfaces have been developed. Most of these systems use glass as a substrate, which has several drawbacks, including high fragility and costs, especially if implemented for fluorescence microscopy. In addition, conventional fabrication technologies such as microcontact printing (µCP) are frequently used for the transfer of biomolecules to the glass surface. In this case, it is challenging to adjust the biomolecule density. Here, we show that cyclic olefin polymer (COP) foils, with their encouraging properties, including the ease of manufacturing, chemical resistance, biocompatibility, low water absorption, and optical clarity, are a promising alternative to glass substrates for the fabrication of micropatterns. Using a photolithography-based approach, we generated streptavidin/biotinylated antibody patterns on COPs with the possibility of adjusting the pattern contrast by varying plasma activation parameters. Our experimental setup was finally successfully implemented for the analysis of PPIs in the membranes of live cells via total internal reflection fluorescence (TIRF) microscopy.

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