An energy based cleavage criterion is defined.
The impact of caspase cleavage was confirmed in a cell based model, showing that predominantly caspase-1-mediated fragmentation of expanded ataxin-3 resulted in increased aggregation and treatment with caspase inhibitors prevented inclusion formation in vitro [ 61].
All four compounds were found to induce apoptosis of HL-60 cells, based on caspase-3 cleavage (Fig. 1), in accordance with the numerous literature reports [8 16, 29, 30].
To exclude this possibility, we used aprotinin, a potent inhibitor of trypsin-like proteinases, in the in vitro and cell-based cleavage tests.
To determine whether transduction of wild-type and mutant ank constructs affected proliferation of transduced cells, we determined the proliferation and cell viability based upon the cleavage of the tetrazolium salt, WST-1, by mitochondrial dehydrogenases in viable cells.
A mammalian cell assay based on endogenous secretase-mediated cleavage of an APP-Gal4-VP16 fusion protein that activates a luciferase reporters gene has been implemented as a HTS screen [38], but has the limitation that more than one endogenous mammalian protease can cleave this engineered substrate.
Transfected cells were maintained for 48 h prior to cell viability assays based on the cleavage of the tetrazolium salt WST-1 (Roche Diagnostics, Germany).
The 10 mg/L of PTEN protein from HighFive cell culture is based on DTT cleavage from this resin to liberate the thioester protein, but is somewhat variable from run to run so we have changed the revised text to reflect that the yield is in the range of 8 10 mg/L.
NSCLC cell proliferation was assessed by using an assay based on cleavage of the tetrazolium salt WST-1 to formazan by cellular mitochondrial dehydrogenases (Roche, Indianapolis, IN) as reported earlier [ 22].
Probe 2 was based on cleavage of amide and photo-induced energy transfer (PET) effect, which was stable, cell-membrance-permeable, low cytotoxic, and highly selective and sensitive for hNQO1.
This XTT cell proliferation assay is based on the cleavage of the yellow 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2 H-tetrazolium-5-carboxanilide inner salt (XTT) by ubiquitous dehydrogenases leading to the formation of an orange formazan dye.
