Cytotoxicity assays

For inhibition of cellular proteases, 30 μM of the SKI-1/S1P inhibitor PF-429242 dihydrochloride (Sigma-Aldrich, Taufenkirchen, Germany) and 100 μM SPP inhibitor (Z-LL)₂ ketone (Merck KGaA, Darmstadt, Germany) were used. Inhibitors were diluted in DMSO as recommended by the manufacturers. Cytotoxicity assays (Promega, Madison, USA) were performed to confirm that the inhibitor concentrations used did not affect cell viability.

Cellular cytotoxicity was evaluated by standard lactate dehydrogenase release assays following the manufacturer’s instructions (Cytotoxicity Assays, Promega, United States). First, we seeded 96-well plates with 1 × 10³ cells. The plates were then incubated for 24 h with sAxl at different levels. Ten microliters of 10 × lysis buffer was added into each well to encourage the maximum release and then incubated for 45 min at 37°C. Next, 50 μl of the supernatant was transferred to a fresh plate, and 50 μl of the substrate was added. The plate was then sealed and incubated for 30 min at room temperature in the dark; 50 μl of stop solution was added to each well to terminate the reaction. The optical density was then read at 490 nm, and the results were calculated by the following formula: