The inhibitors were added to the cultured cells from stock solutions in DMSO to the desired final concentration. For cytotoxicity assays, HeLa cells were grown in 96-well plates (20,000–30,000 cells per well) and incubated with the test compounds for 3 days before cell viability was evaluated with the help of a tetrazolium dye assay (XTT assay, AppliChem GmbH, Darmstadt, Germany).
Inhibitor assays of endogenous DYRK1A activity were performed with overexpressed GFP-SF3B1-NT as described previously [21 (link)]. Briefly, HeLa cells were grown in 6-well plates and treated with test compounds for 18 h before cells were lysed with 100 μL SDS lysis buffer (20 mM Tris HCl pH 7.4, 1% SDS) at 96 °C and sonicated. Total cellular lysates were analysed by immunoblotting with a custom-made rabbit antibody for detecting phosphorylated T434 [43 (link)] and a goat antibody for GFP (no. 600-101-215, Rockland Immunochemicals, Gilbertsville, PA, USA). pT434 signals were quantified using the AIDA Image Analyzer 5.0 program (Raytest, Straubenhardt, Germany). Relative phosphorylation of SF3B1 was calculated by normalisation to total protein levels as determined from GFP immunoreactivity. IC50 values were determined by non-linear curve fitting using the GraphPad Prism 5.0 program (GraphPad Software, La Jolla, CA, USA).
Inhibitor assays of endogenous DYRK1A activity were performed with overexpressed GFP-SF3B1-NT as described previously [21 (link)]. Briefly, HeLa cells were grown in 6-well plates and treated with test compounds for 18 h before cells were lysed with 100 μL SDS lysis buffer (20 mM Tris HCl pH 7.4, 1% SDS) at 96 °C and sonicated. Total cellular lysates were analysed by immunoblotting with a custom-made rabbit antibody for detecting phosphorylated T434 [43 (link)] and a goat antibody for GFP (no. 600-101-215, Rockland Immunochemicals, Gilbertsville, PA, USA). pT434 signals were quantified using the AIDA Image Analyzer 5.0 program (Raytest, Straubenhardt, Germany). Relative phosphorylation of SF3B1 was calculated by normalisation to total protein levels as determined from GFP immunoreactivity. IC50 values were determined by non-linear curve fitting using the GraphPad Prism 5.0 program (GraphPad Software, La Jolla, CA, USA).
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