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)
(), recN+IAPP
(50 M)
(), recIAPP (50 M)
(), and recIAPP+C (50 M)
(), all
supplemented with preformed IAPP fibrils at a concentration of 30 nM or
incubated with preformed IAPP fibrils (30 nM) alone (),
or staurosporine (2 M)
(). 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 (), 54% for recN+IAPP
(), 28% for
recIAPP (),
and 41% for recIAPP+C ().
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
(), recN+IAPP
(), recIAPP
(), and recIAPP+C
() and control incubation with buffer alone
().
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
(), recN+IAPP
(), recIAPP
(), and recIAPP+C
() and
control incubation with buffer alone (). 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 .