Figure 2
Bacteria-delivered enzyme prodrug cytotoxicity assay. Calculated IC50 of HCT-116 cells post-incubation with E. coli cells (over-expressing either NfsA_Ec, NfsB_Ec, NfsB_Pa, or an empty pUCX control) across a serial 2-fold dilution series of A. CB1954 (25 μM to 400 μM); or B. nitro-CBI-DEI (0.06 μM to 15 μM). Following 4 h of incubation bacteria and prodrug were removed by PBS washes, and surviving HCT-116 cells allowed to recover in fresh Dulbecco's modified Eagle's medium supplemented with fetal calf serum, 10 mM HEPES and 100 μg/ml chloramphenicol, as described [8]. Percentage cell survival at each prodrug concentration was then calculated relative to an unchallenged control by CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data are the mean of three independent experiments, each performed in duplicate; and error bars are ± 1 standard deviation. ** indicates p < 0.005 and *** p < 0.001 by one-way ANOVA with Dunnett comparison of test to control.