Date of download: 5/28/2016 Copyright © 2016 SPIE. All rights reserved. Schematic representation of the biosynthetic FRET probes. The linker of CD2 is 17-amino-acid peptide MHDQLTEEVDVAD ̱ GSEL, which contains a caspase-2 recognition site sequence. It is different from CD3 (a 16-amino-acid peptide MHDQLTEEDEVD ̱ GSEL with a linker containing a caspase-2 recognition site sequence). (The cleavage site is indicated by an underline.) Figure Legend: From: Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis J. Biomed. Opt. 2006;11(2): doi: /

Date of download: 5/28/2016 Copyright © 2016 SPIE. All rights reserved. Characterization of the FRET probes. (a) The emission spectra were measured using a spectrofluorometer (excitation wavelength=433nm) while the recombinant CD2 protein was incubated with caspase-2 (final concentration=2unit∕ml) for 1.5h. (b) The emission spectra were measured using a spectrofluorometer (excitation wavelength=433nm) while the recombinant CD3 protein was incubated with caspase-3 (final concentration=2unit∕ml) for 1h. (c) Western blot analysis of the recombinant CD2 and CD3 proteins exposed by activated caspases; the purified CD2 protein was incubated with caspase-3 (lane 1) and caspase-2 (lane 2) for 1.5h, and the purified CD3 protein was incubated with caspase-2 (lane 3) and caspase-3 (lane 4) for 1.5h (final concentration=2unit∕ml). Figure Legend: From: Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis J. Biomed. Opt. 2006;11(2): doi: /

Date of download: 5/28/2016 Copyright © 2016 SPIE. All rights reserved. Properties of CD2 and CD3 in cisplatin-induced apoptosis. (a) HeLa cells that stably expressed the CD2 fusion proteins were incubated with cisplatin (10μg∕ml) for 7to12h. The cell suspensions were then subjected to spectral analysis with excitation wavelength at 433nm. (b) HeLa cells that stably expressed the CD3 fusion proteins were incubated with cisplatin (10μg∕ml) for 7to12h. The cell suspensions were then subjected to spectral analysis with excitation wavelength at 433nm. (c) Cleavage assay of CD2 in lysates, prepared from cisplatin-treated HeLa cells which stably expressed CD2; its cleavage was then examined by Western blotting using an anti-GFP antibody. Lane 1, the lysates were prepared from cisplatin-treated HeLa cells. Lane 2, the lysates were prepared from cisplatin-untreated HeLa cells. Lane 3, the lysates were prepared from the CFP expressing HeLa cells. (d) Cleavage assay of CD3 in lysates, prepared from cisplatin-treated HeLa cells that stably expressed CD3; its cleavage was then examined by Western blotting using an anti-GFP antibody. Lane 1, the lysates were prepared from cisplatin-treated HeLa cells. Lane 2, the lysates were prepared from cisplatin-untreated HeLa cells. Figure Legend: From: Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis J. Biomed. Opt. 2006;11(2): doi: /

Date of download: 5/28/2016 Copyright © 2016 SPIE. All rights reserved. Single-cell imaging FRET changes of CD2 in response to caspase activation during cisplatin-induced apoptosis in living HeLa cells. (a) Ratio of FRET images of the CD2 expressing HeLa cells. HeLa cells that stably expressed the CD2 fusion proteins were cultured in a 96-well plate for 24h; imaging analysis was started immediately after exposure to cisplatin (30μg∕ml). Scale bar, 20μm. (b) Dynamics of activation of caspase-2 in individual HeLa cells during cisplatin-induced apoptosis as examined in (a). The values of RFRET are from the ROI, which is indicated by a circle. Figure Legend: From: Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis J. Biomed. Opt. 2006;11(2): doi: /

Date of download: 5/28/2016 Copyright © 2016 SPIE. All rights reserved. Single-cell imaging FRET changes of CD3 in response to caspase activation during cisplatin-induced apoptosis in living HeLa cells. (a) Ratio of FRET images of the CD3 expressing cells; HeLa cells that stably expressed the CD3 fusion proteins were cultured in a 96-well plate for 24h; imaging analysis was started 40min after exposure to cisplatin (30μg∕ml). Scale bar, 20μm. (b) Dynamics of activation of caspase-3 in individual HeLa cells during cisplatin-induced apoptosis as examined in (a). The values of RFRET are from the ROI, which is indicated by a circle. Figure Legend: From: Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis J. Biomed. Opt. 2006;11(2): doi: /