Figure 3
From: Anchored FRET sensors detect local caspase activation prior to neuronal degeneration
Monitoring of FRET sensor molecule cleavage in neuronal cells undergoing apopotosis. Transfected and differentiated SH-SY5Y cells were treated with 10 μM staurosporine to induce apoptosis. (A) FRET intensity time-lapse imaging of FRET sensors. Time is indicated on top. The frame before the initial drop in FRET intensity was chosen as zero time point for the caspase-3 (DEVD) and -9 (LEHD) sensors, whereas initiation of neurite retraction was chosen as zero time point for the control (LEVA) sensor. Corresponding graphs show the quantitative change in FRET intensity. The FRET signal decreased in cells expressing the caspase-3 (DEVD), or -9 (LEHD) sensors, but not in cells expressing the control (LEVA) sensor. The gray dashed curve corresponds to the right cell (#1), which has a different zero time point than the left cell (#2) in the LEHD time-lapse experiment. (B) Western blot analysis using anti-GFP antibodies of FRET sensor molecules in extracts of cells expressing the caspase-3 (DEVD) sensor or the uncleavable control (LEVA) sensor incubated for 3 hours in the absence or presence of 10 μM staurosporine.