Methodology Report

Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation

Figure 5

FRET assays of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C. (a) B-TC-6 cells expressing pFRET2-DEVD were incubated with solubilised recombinant peptides recproIAPP (50  M) (Purple-triangle), recN+IAPP (50  M) (Yellow-square), recIAPP (50  M) (Green-square), and recIAPP+C (50  M) (Blue-bullet), all supplemented with preformed IAPP fibrils at a concentration of 30 nM or incubated with preformed IAPP fibrils (30 nM) alone (Blue-square), or staurosporine (2  M) (Red-bullet). The FRET ratio at 535/480 nm was determined hourly over 12 hours. Data presented are mean value ±SEM . (b) Cell viability after incubation for 12 hours with solubilised recombinant peptides supplemented with preformed IAPP fibrils (30 nM). Data are presented as mean value ±SEM . Relative to the control, cell viability was determined to 51% for recproIAPP (Purple-triangle), 54% for recN+IAPP (Yellow-square), 28% for recIAPP (Green-square), and 41% for recIAPP+C (Blue-bullet). Compared to the control, a significant difference in the FRET ratio was observed for all four recombinant peptides in an ANOVA test . (c) FRET ratio of pFRET2-DEVD expressing B-TC-6 cells after 12 hours of incubation with recombinant amyloid-like fibrils corresponding to 50  M. RecproIAPP (Purple-triangle), recN+IAPP (Yellow-square), recIAPP (Green-square), and recIAPP+C (Blue-bullet) and control incubation with buffer alone (Blue-square). Relative to the control, cell viability after incubation with recproIAPP was 85%, with recN+IAPP 102%, with recIAPP 104% and with recIAPP+C 89%. No statistically difference between the negative control and the four recombinant peptides was observed in an ANOVA test . (d) Incubation of pFRET2-DEVD expressing B-TC-6 cells with recombinant peptides in solubilised form (50  M). RecproIAPP (Purple-triangle), recN+IAPP (Yellow-square), recIAPP (Green-square), and recIAPP+C (Blue-bullet) and control incubation with buffer alone (Blue-square). Relative to the control, cell viability after 12 hours incubation with recproIAPP was 86%, recN+IAPP 97%, recIAPP 102%, and recIAPP+C 89%. The four recombinant peptides did not differ significantly from the negative control in an ANOVA test .
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