DEVD‐NucView488: a novel class of enzyme substrates for real‐time detection of caspase‐3 activity in live cells

Abstract

Live-cell detection of intracellular enzyme activity requires that substrates are cell-permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real-time detection of apoptosis in living cells. We have synthesized a highly cell-permeable caspase-3 substrate, DEVD-NucView488, by linking a fluorogenic DNA-binding dye to the caspase-3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD-NucView488 detected caspase-3 activation within a live-cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD-NucView488 exhibited no toxicity and normal apoptotic progression. DEVD-NucView488 is an ideal substrate for kinetic studies of caspase-3 activation because it detects caspase-3 activity in real-time and also efficiently labels DNA in nuclei of caspase-3-activated cells for real-time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real-time intracellular enzyme activity.

Recurrent ubiquitin B silencing in gynecological cancers establishes dependence on ubiquitin C

“…To assess cellular outcomes resulting from UBC knockdown, we followed cells in a time course after siRNA transfection to better understand if cells were undergoing a cell cycle arrest or a cell death response. The cellular response was observed using real-time microscopy with the Incucyte apparatus that allowed us to look at cell morphology and, in combination with a fluorogenic caspase substrate dye (24), allowed us to additionally monitor the induction of caspases involved in executing the apoptotic program ( Figure 4). UBB LO (OVCAR8) and UBB WT (OC316) cells transfected with siRNA targeting PLK1 show a morphology, previ-( Figure 2A).…”

“…mRNA levels for UBB and UBC were assessed 24 hours after transfection. Caspase-3/7 was detected using an IncuCyte Green Apoptosis assay 4440 (Essen Biosciences) (24).…”

Transient P2X7 Receptor Activation Triggers Macrophage Death Independent of Toll-like Receptors 2 and 4, Caspase-1, and Pannexin-1 Proteins

“…Following enzymatic cleavage, the released NucView488 moiety becomes green fluorescent upon binding to DNA (28). In selected experiments, macrophages were coloaded with TMRE by incubating the cells with 200 nM TMRE (diluted from a 20 mM stock solution in DMSO) for 5 min at 37°C.…”

The microtubule poison vinorelbine kills cells independently of mitotic arrest and targets cells lacking the APC tumour suppressor more effectively

“…S1B,C). To identify apoptotic cells, we used Nucview, a fluorescent reporter for active caspase-3 (Cen et al, 2008). In parallel, we used mCherry-labeled lamin B1 to report on the integrity of the nuclear envelope as a marker for interphase cells (Liu et al, 2003).…”

“…For live-cell fluorescence markers, U2OS cells were co-transfected with 0.2 mg DNA encoding mCherry-tagged lamin B1 along with 10 nM APC and/or p53 siRNAs (Dharmacon, Qiagen) using Fugene 6 (Roche) according to the manufacturer's instructions. NucView (Biotium) was added to medium to detect caspase-3 activation as described previously (Cen et al, 2008). To induce mitotic arrest and cell death, U2OS cells were treated with 1.5 mg/ml nocodazole or various concentrations of vinorelbine (see figure legends).…”