Figure 2.

In vitro effects of secreted proteins from transformed S. typhimurium on 4T1 mammary carcinoma cells. Filtered supernatants were acquired from VNP pRA-ZsG (ZsGreen sup.) and VNP pRA-TR (TRAIL sup.) after growth for 4 h in minimal medium after induction with 5 J m⁻² UV irradiation. Control supernatants were acquired from sterile medium. (A) Caspase activity assays were conducted on 4T1 cells after application of experimental supernatants or recombinant mouse TNF-α at 50 ng ml⁻¹ for 24 h. Significant increases in caspase-3 and caspase-8 activities were observed after treatments with TNF-α and VNP pRA-TR supernatants when compared with controls (^*P<0.05). Addition of the caspase-3 inhibitor, DEVD-fmk, significantly reduced activity. (B, C) Flow cytometry for annexin-V-FITC and propidium iodide was conducted on 10 000 4T1 cells per treatment in triplicate after application of experimental supernatants or TNF-α at 50 ng ml⁻¹ for 48 h. (B) Flow cytometry dot plots after treatment with control (left) and VNP pRA-TR supernatants (right) indicate cell death (annexin-V and propidium iodide positive) proportions of 5.7 and 12.6%, respectively. (C) Results of flow cytometry indicate cell fractions undergoing cell death (annexin-V positive, propidium iodide positive) and early apoptosis (annexin-V positive, propidium iodide negative). Significant increases in cell death and early apoptosis were observed in 4T1 cells after treatment with the VNP pRA-TR supernatant (^*P<0.05).