Hygromycin b

Human osteosarcoma U2OS cells expressing the tetracycline responsible element of Flp-In™ T-REx system (FT-U2OS, gift from Karmella Haynes, Arizona State University, (Haynes and Silver, 2011 (link))) were cultured in Dulbecco’s Modified Eagle’s medium without sodium pyruvate, L-Arg, and L-Lys (DMEM, Wisent) supplemented with 10% of 10 kDa cut-off fetal bovine serum (Wisent), 100 μg/mL streptomycin, 100 units/mL penicillin (Thermo), 15 μg/mL blasticidin (Wisent), and 150 μg/mL hygromycin B (Wisent). The cell media was also supplemented with 0.398 mM L-Arg, 0.274 mM L-Lys, and 3.47 mM L-Pro. The cells were grown at 37°C with 5% CO₂ in 10 cm dishes (TPP, FroggaBio) or in 6-well plates (Greiner BioOne). Following the recommendations of Flp-In™ T-REx cell line development of ThermoFisher Scientific (http://www.thermofisher.com/), we developed cell lines stably expressing the wild-type CSNK2A1-HA (WT) or the inhibitor resistant forms of double mutant (DM, V66A/I174A) of the kinase with tight tetracycline regulation. The exogenous CSNK2A1 has a C-terminal HA tag to be able to distinguish from the endogenous kinase. The cell lines were induced 48h prior to inhibitor treatment with 1 μg/mL tetracycline and were kept induced during the inhibitor treatment. Cells were challenged in two independent experiment with a range of inhibitor concentrations as indicated in the figures.

Two libraries were employed for the DHFR PCA screen: a library with C-terminal DHFR F[1,2] tag and a library with C-terminal DHFR F[3] tag (Tarassov et al., 2008 (link)). A query strain was taken out from each library with Slt2 tagged with either fragment and mated against the opposite libraries by overnight incubation on YPD. After mating, diploid cells were selected for by incubation for 2 days on YPD medium with 100 μg/ml nourseothricin (Werner Bioagents, Jena, Germany) and 250 μg/ml hygromycin B (Wisent Bioproducts, Saint-Jean-Baptiste, Canada). This step was repeated once. Next, the strains were transferred to synthetic complete medium (4% (w/v) Noble agar) with 200 μg/ml methotrexate (Bioshop Canada, Montréal, Canada) and without adenine or ammonium sulfate. Pictures of the strains were taken after 4 days incubation at 30°C. Colony size was analyzed using the Balony software (Young and Loewen, 2013 (link)).