Countess 2 fl automated counter

Transwell migration assays were performed using the CytoSelect 24-well Haptotaxis Assay and the MCPyV-positive MCC PeTa cell line. Briefly, 1 × 10⁶cells/ml were resuspended in serum depleted (0.5% (v/v) FBS) medium containing inhibitor or vehicle control and incubated for 24 h. 300 µl medium containing inhibitor and cells was added to the upper chamber and 500 µl of serum enriched (20% (v/v) FBS) growth medium was added to the lower chamber of the CytoSelect plate before incubation for 48 h. Cells in each chamber were resuspended by pipetting and aliquots mixed with trypan blue before quantification of cell number using a Countess II FL Automated Counter (Thermo Fisher). The percentage of migrated cells for each condition was calculated before comparison to an untreated control.

Cell lines were obtained from sources indicated in Supplementary Table 1. All human cell lines were verified by the supplier; if we had no record of this, we independently verified them using short tandem repeat (STR) profiling (Eurofins). Prior to use, all cells tested negative for mycoplasma using an e-Myco Plus kit (Intron Biotechnology). Cells were grown to 60–100% confluence and harvested by trypsinisation and counted using a Countess II FL automated counter (Thermo Fisher Scientific)). Cell lines with large cells were manually counted with a haemocytometer. Pellets corresponding to 1.4–70 × 10⁶ cells were washed twice with ice cold PBS by centrifugation, snap frozen using liquid nitrogen and stored at −80 °C. Pellets were thawed on ice and lysed in NP40 lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% NP40 substitute, 1/250 mammalian protease inhibitor cocktail (Sigma)). Lysate protein concentration was determined using Pierce bicinchoninic acid (BCA) assay. n = 3 independently prepared cell pellets were used for PSAQ analysis.