Incucyte zoom time lapse imaging system

To monitor virus infection and spread, S462TY or SK-N-AS cells were seeded at 2,000 cells per well in a 96 flat bottom well plate and allowed to attach overnight. Cells were washed with sterile PBS, then either fresh media or conditioned media (prepared from virus-infected cells, see above) was then added to each well and treated with or without HSV1716-GFP (MOI = 0.5) in the presence or absence of alisertib (50 nM). Virus infection was monitored by phase contrast and GFP-imaging every three hours using the IncuCyte Zoom time lapse imaging system (Essen Biosciences, Ann Arbor, MI, USA).

To determine two-drug synergy in vitro, cells were seeded at 2,000 cells per well in a 96 flat bottom well plate and allowed to attach overnight. Cells were then washed with sterile PBS. Fresh media was added containing HSV1716-GFP (MOI = 0, 0.01, 0.1, 1, 10) alone, alisertib (0, 15, 31, 62, 125, 250, 500, 1000 nM) alone or the combination of the HSV176-GFP and alisertib at the various MOI and concentrations, respectively. Cell proliferation was monitored by phase contrast imaging every 3 hours using the IncuCyte Zoom time lapse imaging system (Essen Biosciences, Ann Arbor, MI, USA). Synergy was determined using the Chou-Talalay method of synergy analysis [54 (link)] using CompuSyn for Drug Combinations Analysis Software (ComboSyn, Inc., Paramus, NJ, USA).