Figure 4.

Analysis of bystander effect and of CPE release via exosomes in optCPE-transfected PC cells. (A) Analysis of cytotoxicity of released optCPE. Media of vector control (VC) or optCPE-transfected MIA PaCa-2 and PA-TU-8902 cells collected at 24 h, 48 h and 72 h were added to respective non-transfected cells. After 72 h cytotoxicity was determined by MTT. Significant reduction of viability in PA-TU-8902 and MIA PaCa-2 cells was induced by supernatants collected 48 h and 72 h after transfection. For PA-TU-8902 cells, higher toxicity was observed than in MIA PaCa-2 cells. This correlated with the released optCPE amount. (B) Western blot of supernatant and exosomes (specific exosomal marker heat shock protein 70, HSP70 and tetraspanin CD63, Figure S7) from transfected MIA PaCa-2 and PA-TU-8902 cells. (C) Quantification of optCPE within supernatant and exosomes of optCPE-transfected MIA PaCa-2 and PA-TU-8902 cells by CPE ELISA revealed concentrations of 324.4 and 382.7 ng mL⁻¹ CPE in supernatant and 242.6 and 344.5 ng mL⁻¹ CPE in exosomes, respectively. Data are represented as mean values ± S.D. (n = 3), expressed as mean percentage of VC. (* p < 0.05; ** p < 0.01; **** p < 0.0001).