Trypan blue

Cells were plated in triplicate or quadruplicate in 96-well plates, drug-treated the following day, and harvested 72–96 h later using CellTiter Glo (see manufacturer’s instructions, Table S1). Trametinib was removed from resistant cells two days prior to plating in order to be able to assess the effects of ABL/DDR inhibitors in the absence or presence of trametinib. To measure trypan blue exclusion, cells were plated in 60 mm dishes, drug-treated for 96 h, trypsinized, and the percentage of live cells were scored by diluting cells 1:1 in trypan blue (Biorad, Hercules, CA, USA) and counting on a TC-20 automated cell counter (Biorad, Hercules, CA, USA).

A 24-well transwell chamber with polycarbonate membrane (5 µm) (catalog no. 3421; Costar, Kennebunk, ME, USA) was pre-coated with fibronectin (10 µg/ml) (F1141-1MG, Sigma-Aldrich). Jurkat cells were pretreated with ITK inhibitors (2 µM) for 24 hours in serum-free RPMI 1640 media. Chemotaxis medium (RPMI 1640 medium containing 25 mM HEPES buffer and 1% BSA) with and without CXCL12 (100 ng/ml) (350-NS-010, R&D Systems, Minneapolis, MN, USA) was added to the lower chamber with ITKi. Jurkat cell suspension (10⁶ cells in 100 µl) were placed in the upper wells. The cells were allowed to migrate for 3 hours at 37 °C. Migrated cells were collected in the lower compartment stained with trypan blue (BioRad) and counted by hemocytometer or automated cell counter.

HeLa cells were grown in Gibco™ DMEM Medium (1x Dulbecco’s Modified Eagle Medium; ThermoFisher Scientific, Cat. No. 31885-023) including 10% Fetal Bovine Serum (Biochrom AG; Cat. No. S0415), 1% MEM NEAA (100x) (Minimum Essential Medium Non-Essential Amino Acid; ThermoFisher Scientific; Cat. No. 11140-035) and 1% Penicillin-Streptomycin (Sigma; Cat. No. P0781) overnight at 37 °C. Jurkat cells are grown in Gibco™ RPMI Medium 1640 (1x) (Thermo Fisher Scientific; Cat. No. 31870-025) including 10% Fetal Bovine Serum (Biochrom AG; Cat. No. S0415), 1% MEM NEAA (100x) (Minimum Essential Medium Non-Essential Amino Acid; ThermoFisher Scientific; Cat. No. 11140-035) and 1% Penicillin-Streptomycin (Sigma; Cat. No. P0781). After aspiration of medium, and PBS wash, HeLa cells are incubated in Trypsin/EDTA (Sigma; Cat. No. T3924) for 5–10 min at 37 °C followed by addition of medium to stop trypsination of cells. After centrifugation (Hettich Rotina 380 R; 5 min × 300 g) of HeLa or Jurkat cells and an additional washing in PBS, cells are counted in Counting Slides (BIO RAD; Cat. No. 145-0011) in TC20 Automated Cell Counter (BIO RAD) by staining with trypan-blue (BIO RAD; Cat. No. 145-0013). In general, a fraction of >80% of viable cells were determined. After counting, cells were diluted to the intended cell number per sample.

100 000 cells were seeded in 6-well plate and were counted using trypan-blue (Bio-Rad) every second day for 6 days.

U2OS and IMR90 cells were seeded 24 hours prior to treatments in 10 cm plates (Corning) at 60% confluence. Cells were then treated with 350 μM (U2OS) or 88 μM (IMR90) TBH (Sigma #458139) for 16 hours. Cell death was assessed by imaging the cells under a white-light microscope and by counting live and dead cells using Trypan-Blue (BioRad) and a cell counter. Supernatants were collected and added to trypsinized cells before counting. All assays were performed at least three times. For IC50 assays, 20 000 IMR90 cells of each condition were plated per well of a 48 well plate. Cells were plated 24 hours prior to TBH treatment. Cells were then treated with either: 0, 10, 20, 40, 60, 80, 90, 100, 120, 160, 180 or 200 μM of TBH for 16 hours. Cells were then fixed with 1% gluteraldehyde in PBS and stained with Crystal Violet (Sigma #C0775). The dye was then resuspended in 10% acetic acid and dosed with a spectrophotometer. GraphPad Prism was used to generate IC50 curves and determine IC50 values.

Different Asia-1 isolates (Asia TUR 6/2014, Asia TUR 6/2014-PT, Asia HKN 5/2005, Asia PAK 5/2012) were selected from archival stocks of the Friedrich-Loeffler-Institut (FLI) and were tested for their ability to successfully infect BHK-2P cells. Virus was first grown on adherent BHK164 cells and the gained supernatant was used to infect the BHK-2P, followed by repeated passaging in BHK-2P. Successful virus replication was defined as a decrease in cell viability and growth. Cell viability was determined using trypan blue (Bio-Rad, Hercules, CA, USA) and an automatic cell counter (Bio-Rad, model TC20™, Munich, Germany). To check the supernatant for remaining infectivity, it was passaged on adherent cells (infection control).
For further experiments, a representative Asia-1 strain, the Shamir/ISR/1989 isolate, was used. As comparison, a second strain, FMDV A₂₄ Cruzeiro, which can infect the adherent and the suspension cell lines, was used in selected experiments. A third strain, FMDV O₁ Manisa, was used in experiments testing the pH sensitivity. All virus strains were used as cell culture supernatant. For a detailed listing of the used isolates and passage histories see the Supplementary Materials (Table S1).