Alectinib

For all experiments involving chemical additives to fly food, the compound was dissolved in a stock solution of either ethanol or DMSO and added to the fly food while it was still liquid but had cooled to 50°C. The stock solution was added to the food, mixed well, dispensed into individual fly vials, and allowed to cool to room temperature overnight before storage at 4°C. On the day of experiments, food vials were warmed to room temperature before being used. For experiments involving the GeneSwitch system, RU‐486 (Sigma, stock solution 100 mM in ethanol) was added to the food at a final concentration of 200 µM, with 2 ml/L ethanol used as the vehicle control condition. For experiments involving Alk inhibitors, stock solutions of Alectinib (MedChem Express), Crizotinib (LKT Labs) or TAE‐684 (Generon) were prepared at 5 mM in DMSO and added to the food at the concentrations indicated in each figure. For all Alk inhibitor experimental and vehicle control conditions, DMSO was added to a final concentration of 0.2%. For all experiments, flies were allowed to develop on control food without any additives and then divided into groups to be put on vehicle‐ or drug‐containing food 2 days after emerging as adults.

Three different inhibitors of SRPK1 and SRPK2 were used to assay effects on viral replication: SRPIN340, SPHINX31, and Alectinib (MedChem Express). SRPIN340 and SPHINX were suspended in DMSO at 1000X the final concentration required, whereas Alectinib was resuspended at 500X. The inhibitors were then diluted to the appropriate concentrations in cell-specific medium. Cells were treated with inhibitor 12 hours before they were infected. Cells were then infected for 1 hour as described earlier. After infection, medium with 2% FBS and either drug or DMSO as a control was added to each well (for qRT-PCR and Calu-3 microscopy assays) or the inoculum was removed and medium with 2% FBS and either drug or DMSO was added to each well (for infectious titer quantification).