Zymoxin-Mediated Removal of HCV Infected Cells Figure 2. Colony formation assay for the assessment of ‘‘mCherry-NS3 activated MazF’’ cytotoxicity toward na¨ıve cells. A day before transfection, 7.56105 HEK293 T-REx cells where seeded per well in 6 wells plate and subsequently transfected with 2 mg of plasmids encoding either mCherry-NS3 activated MazF, mCherry (only the fluorescent protein) or EGFP- MazF (where MazF is not fused to its inhibitory peptide). 48 hours later, transfection efficiency was assessed by fluorescence microscopy and was determined as equal between the plasmids. Transfected cells were than trypsinized, counted and seeded in 3 fold dilutions (starting from 150,000 cells/well) in 6 well plates and were incubated for 10 days in the presence of 1 mg/ml of G418 (to which all three plasmids confer resistance). Surviving colonies were fixed and stained with Giemsa (upper panel). Number of surviving Colonies from wells that were seeded with 5556 cells was determined by manual counting. Each bar represents the mean 6 standard deviation (SD) of a set of data from two wells (lower panel). doi:10.1371/journal.pone.0032320.g002 In support of these findings, an immunoblot assay revealed a near complete cleavage of the zymoxin following tetracycline-induced expression of NS3 protease in NS3-activated MazF expressing cells (Figure 4, lower panel). Presumably, this proteolytic activation of the ribonuclease toxin resulted in a cessation of cellular protein synthesis soon after induction, what explains the detection of relatively low levels of NS3 protease in these cells. As expected, no zymoxin cleavage could be detected in cells expressing the uncleavable form of the toxin. A faint band, corresponding to a cleaved zymoxin, can also be detected in uninduced cells that express the NS3 cleavable construct. This cleavage can be attributed to the ‘‘leakiness’’ of the Tet inducible system, allowing a very low PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32320