Figure 4

Bystander effect from metabolic activation of nitro-CBI-DEI in multicellular layer cultures. Clonogenic survival curves were generated using cells from three-dimensional multicellular layers exposed to nitro-CBI-DEI under hyperoxic conditions (95% O₂; used to avoid hypoxia/necrosis in cells at the centre together with confounding activation of nitro-CBI-DEI due to hypoxia). Multicellular layers consisted of 100% HCT116 wild type ‘target’ cells or cocultures of target cells in the presence of a small percentage of HCT116 NfsB_Pa ‘activator’ cells (5.8 ± 3.2%). To establish the C₁₀ values of target and activator cells in co-culture, cells were plated in both non-selective and selective media (the latter amended with 3 μM puromycin). The target cell population was calculated by subtracting the number of puromycin resistant activator cells (colonies counted on selective plates) from the total cell population (colonies counted on non-selective plates). Points are the combined data sets from two independent experiments with overlapping data points; and error bars are ± 1 standard deviation. The dashed line represents one log of cell kill. The C₁₀ values in targets alone (T), targets in co-culture (T_C) or activators in co-culture (A_C) were interpolated.