ITRACONAZOLE INHIBITS ENTEROVIRUS REPLICATION BY TARGETING THE OXYSTEROL-BINDING PROTEIN

Strating, Jeroen R.P.M.; van der Linden, Lonneke; Albulescu, Lucian; Bigay, Joëlle; Arita, Minetaro; Delang, Leen; Leyssen, Pieter; van der Schaar, Hilde M.; Lanke, Kjerstin H.W.; Thibaut, Hendrik Jan; Ulferts, Rachel; Drin, Guillaume; Schlinck, Nina; Wubbolts, Richard W.; Sever, Navdar; Head, Sarah A.; Liu, Jun O.; Beachy, Philip A.; De Matteis, Maria A.; Shair, Matthew D.; Olkkonen, Vesa M.; Neyts, Johan; van Kuppeveld, Frank J.M.

For live-cell imaging experiments HeLa R19 cells were seeded in compartmentalized CELLview petridishes (Greiner Bio-One) and transfected O/N with pEGFP-hOSBP. Dishes were transferred to a humidified, CO 2 -and temperature-controlled chamber (Tokai-Hit) for imaging on a Nikon A1R confocal laser scanning microscope mounted on a Nikon Eclipse-Ti base, cells were selected for imaging and the reference image (t=0) was taken. For the long-term imaging experiment, compounds were added from a two-fold concentrated dilution to the compartments and cells at four different positions per well were imaged O/N. From 0 to 30 min after addition of the drugs, images were taken as fast as possible (i.e. at ~1.5 min intervals), then intervals were gradually increased to 15 min intervals from 2 hr after addition of the compounds onward to prevent bleaching and phototoxicity during the O/N imaging: from 30 to 60 min intervals were 5 min, until 3.5 hr intervals were 15 min and finally intervals were 30 min for the rest of the experiment. Images were processed and quantified using the Nikon NIS-Elements software. For quantification, regions of interest were defined in the perinuclear region where a stronger OSBP signal was observed than in the rest of the cytoplasm (i.e. the Golgi) and the change in average fluorescence intensity in this area was quantified. A movie was assembled using Adobe Premier Pro CS6 software.