Real-Time Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation
Figure 2
Parallel performed ThT assay, electron
microscopy, and FRET assay of synthetic IAPP peptide.
(a) ThT
assay of synthetic IAPP peptide (50 M) seeded with preformed IAPP fibrils
(30 nM)
(), ThT assay buffer seeded with preformed
IAPP fibrils (30 nm) (), and ThT assay
buffer (). Data presented are mean values ±SEM . Synthetic IAPP peptide
(50 M) seeded with
preformed IAPP fibrils (30 nM) incubated for (b) 2 hours of 50 M,
(c) for 4 hours, (d) and for 12 hours; all
samples were negative contrasted. In (b), the higher magnification shows small
prefibrillar ring shaped structures. In (c), thin thread-like protofibrils are
visible and in (d) more mature amyloid-like fibrils are apparent. Large-scale
bars represent 100 nm, and the smaller-scale bar in (b) represents 20 nm. (e) FRET ratio of 535 nm/480 nm measurements of KRHG buffer with seeds of preformed
IAPP fibrils (30 nM) (), synthetic
IAPP peptide (50 M) seeded with preformed IAPP fibrils
(30 nM) (), and staurosporine
(2 M)
(). Measurements were performed during a 12-hour
period with individual measurements each hour. Data presented are mean values ±SEM .
(f) FRET ratio after 12 hours of measurements. Staurosporine sample was set as zero
and was subtracted from assay measurements, and the negative control was set as
100% viable cells (blue column). The FRET ratio of the B-TC-6 cells incubated
in synthetic IAPP peptide (50 M) seeded with preformed IAPP fibrils
(30 nM) was
37% of the negative control corresponding to a population of 65% apoptotic
cells (green column). Data presented are mean values ±SEM . The difference between the negative control
and IAPP was considered significant in a two-tailed unpaired t-test .
), ThT assay buffer seeded with preformed
IAPP fibrils (30 nm) (
), and ThT assay
buffer (
). Data presented are mean values ±SEM . Synthetic IAPP peptide
(50 M) seeded with
preformed IAPP fibrils (30 nM) incubated for (b) 2 hours of 50 M,
(c) for 4 hours, (d) and for 12 hours; all
samples were negative contrasted. In (b), the higher magnification shows small
prefibrillar ring shaped structures. In (c), thin thread-like protofibrils are
visible and in (d) more mature amyloid-like fibrils are apparent. Large-scale
bars represent 100 nm, and the smaller-scale bar in (b) represents 20 nm. (e) FRET ratio of 535 nm/480 nm measurements of KRHG buffer with seeds of preformed
IAPP fibrils (30 nM) (
), synthetic
IAPP peptide (50 M) seeded with preformed IAPP fibrils
(30 nM) (
). Measurements were performed during a 12-hour
period with individual measurements each hour. Data presented are mean values ±SEM .
(f) FRET ratio after 12 hours of measurements. Staurosporine sample was set as zero
and was subtracted from assay measurements, and the negative control was set as
100% viable cells (blue column). The FRET ratio of the B-TC-6 cells incubated
in synthetic IAPP peptide (50 M) seeded with preformed IAPP fibrils
(30 nM) was
37% of the negative control corresponding to a population of 65% apoptotic
cells (green column). Data presented are mean values ±SEM . The difference between the negative control
and IAPP was considered significant in a two-tailed unpaired t-test .
