Journal: PLoS ONE

Article Title: Imaging Proteolytic Activity in Live Cells and Animal Models

doi: 10.1371/journal.pone.0066248

Figure Lengend Snippet: A) Schematic of the Caspase 3/7 GloSensor reporter containing an N-terminus coding for the C-Luc domain (358–544) of luciferase and a C-terminus coding for the N-Luc domain (4–354) of luciferase and a adjoining sequence, DEVD, the Caspase 3/7 recognition sequence. B) The functional basis of the reporter, wherein Caspase 3/7 mediated cleavage at the DEVD sequence results in release of the luciferase peptides and reconstitution of the enzymatic activity and an increase in luminescence signal. C) Bioluminescence analysis of cells treated with 200 ng/ml TRAIL. Data is plotted as fold induction standardized to values obtained from vehicle treated cells. D) Western blot for Caspase 3 cleavage using D54 cells treated with TRAIL for 6 hrs. β-Actin was used to standardize loading. E) Bioluminescence analysis of D54 cells treated with varying concentrations (25–100 ng/ml) of an agonist anti-Fas antibody. Data is plotted as fold induction over values obtained from vehicle treated cells at every hour. F) Bioluminescence analysis of cells treated with a pan-Caspase inhibitor Z-VAD (20 µM), 50 µM Docetaxel or with Z-VAD and Docetaxel combined. Data is plotted as fold induction. Experiments were performed at least in triplicates and mean values were plotted ± SEM.

Article Snippet: Live-cell bioluminescent imaging was performed by adding 100 μg/ml of GloSensor cAMP reagent (Promega, WI) to the assay media.

Techniques: Luciferase, Sequencing, Functional Assay, Activity Assay, Western Blot

Journal: PLoS ONE

Article Title: Imaging Proteolytic Activity in Live Cells and Animal Models

doi: 10.1371/journal.pone.0066248

Figure Lengend Snippet: A) Schematic of the pCLEX Caspase 3/7 GloSensor transgene construct. B) Excision of the floxed EGFP-stop cassette when crossed with a Cre expressing mouse strain should result in tissue specific transcription of the reporter. C) Representative bioluminescence images of bi-transgenic (for the reporter and p48-Cre) or mono-transgenic (transgenic for the reporter in the absence of Cre) animals pre- and 30 hrs post-cerulein injection (75 ug/kg, total of 12 injections in 48 hrs). D) Quantification of BLI signal induction upon cerulein treatment. E) Representative bioluminescent and fluorescent (EGFP) ex-vivo images of pancreata from mono- or bi-transgenic animals. F) Representative bioluminescence images of bi-transgenic (right) or mono-transgic (left) animals.

Article Snippet: Live-cell bioluminescent imaging was performed by adding 100 μg/ml of GloSensor cAMP reagent (Promega, WI) to the assay media.

Techniques: Construct, Expressing, Transgenic Assay, Injection, Ex Vivo

Journal: PLoS ONE

Article Title: Imaging Proteolytic Activity in Live Cells and Animal Models

doi: 10.1371/journal.pone.0066248

Figure Lengend Snippet: Glosensor expressing 1833 or D54 cells (A and B respectively) were used to screen a library of 1,280 pharmacologically active compounds (LOPAC). Fold induction of bioluminescence signal intensity over values obtained from vehicle treated cells was plotted at maximal induction (mean ± SEM).

Article Snippet: Live-cell bioluminescent imaging was performed by adding 100 μg/ml of GloSensor cAMP reagent (Promega, WI) to the assay media.

Techniques: Expressing