Rhgas6

Cells cultured in 96-, 24-, or 6-well plates were infected with ZIKV at different MOIs for 1 h at 37°C. The HTO were infected at 10⁵ PFU as described previously (38 (link)). For Axl receptor blockade, SC were preincubated with 10 μg/ml anti-Axl antibody (catalog no. AF154; R&D Systems) or 50 μg/ml anti-Gas6 antibody (catalog no. AB885; R&D Systems), or goat polyclonal IgG control (catalog no. AB-108-C; R&D Systems) for 2 h at 37°C prior to infection. Following infection, wells were replenished with fresh media without antibody. For Gas6 depletion, SC were serum deprived for 3 h prior to infection, infected without serum or in the presence of 5 nM recombinant human Gas6 (rhGas6) (catalog no. 885-GSB; R&D Systems), and then replenished with media containing 5% fetal bovine serum (FBS) following 1-h infection. For Axl kinase inhibition experiments, SC or HTO were preincubated with 1 μM R428 (catalog no. BGB324; Selleckchem) for 2 h prior to infection and after infection replenished with fresh media containing 1 μM R428 or DMSO vehicle control (1:10,000 dilution). ZIKV titers in the culture supernatant and intracellular ZIKV RNA was quantified by plaque assay and quantitative reverse transcription-PCR (qRT-PCR), respectively, as reported previously (48 (link), 49 (link)).

Human colorectal cancer cells (HCT116, SW480 and SW620) harvested from subconfluent cultures were seeded at 5.000 cells/well in 96-well microplates in medium with 1% FCS and incubated with different concentrations and combinations of rhGas6 (0–100 ng/ml; R&D Systems), hProteinS (0–20 μg/ml; Enzyme Research Laboratories) and 5-FU (0–5 μM). After 48 hours treatment, proliferation was measured using the Cell Proliferation Reagent WST-1 (Roche) and normalized to the signal obtained when using a standard row of cells, seeded at defined densities according to the company's manual.

Intracerebroventricular (ICV) injections into tadpoles were performed using a modified glass capillary tube and Nanoject III programmable nanoliter injector (Drummond Scientific, Pennsylvania, USA). Tadpoles were placed onto a damp paper towel and a small hole was punctured into the head using a modified Pasteur pipette and a small piece of wire. Tadpoles received a 25 nl volume of rhGAS6 (1 µg/ml, R&D Systems) in 3% D-(+)-glucose solution, or saline control, injected at a rate of 100 nl/s. Injection solutions were mixed with a small amount of Fast Green FCF dye to visualise injection into the ventricle. After injection, animals were returned to aquaria.