Zymoxin-Mediated Removal of HCV Infected Cells Figure 3. Cellular colocalization of NS3 protease and the ER-membrane targeted MazF based zymoxin. 16105 Tet-NS3/activated MazF or Tet-NS3/uncleavable MazF cells were seeded on poly-L-lysine coated cover-slips in a 24 well-plate. 12 h later, the cells were supplemented with 1 mg/ml of tetracycline or left untreated. After 24 h, the cells were fixed. Uninduced Tet-NS3/activated (A) and uncleavable (B) MazF cells were permeabilized and incubated with rabbit-polyclonal anti-calnexin antibody followed by Cy2-conjugated anti-rabbit IgG secondary antibody for ER visualization (green). Nuclei of the uninduced cells (A and B) and induced Tet-NS3/uncleavable MaF cells (C and D) were stained by Hoechst 33258 (blue). Slides were then mounted and examined by confocal fluorescence microscope. The bar represents 20 mm. doi:10.1371/journal.pone.0032320.g003 basal transcription of the protease in the absence of externally added tetracycline. Such cleavage could not be detected in ‘‘na¨ıve’’ cells following transfection with NS3-cleavage substrate encoding transgene ([10], data not shown). Apparently, this very low, basal proteolytic activity is well tolerated by NS3 activated zymoxin expressing cells that shows no indication of de-novo protein synthesis inhibition in comparison to cells that express the uncleavable toxin (see CPM values in the upper panel of Figure 4). PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e32320