E) Bioluminescence microscopy of A549-BTR/SBE-NL cells treated as described for panel D.What challenges, if any, would you like to see addressed?

D) Graph shows mean values +SEM for NL bioluminescence in cells treated with 10 ng/ml TGF-β for 22 hours without or with increasing concentrations of SD208 (n = 4 per condition). Graph shows mean values + SEM (n = 4 per condition). C) Stably transfected A549-BTR/SBE-NL cells were treated for 22 hours with increasing concentrations of TGF-β before quantifying NL bioluminescence. Band intensities quantified by ImageJ were normalized to GAPDH, and values under the blot show induction relative to mock treatment. Inset shows bioluminescence image of the 12 well plate and Western blot for levels of NL protein under various treatment conditions. Graph shows mean values ± SEM for firefly luciferase activity (n = 3 per condition). B) A549 cells stably expressing a FL reporter for kinase activity of the type I receptor for TGF-β (BTR) were transiently transfected with SBE-NL and treated with 10 ng/ml TGF-β in the absence or presence of 10 μM of the TGF-βR1 kinase inhibitors SB431542 or SD208 for 22 hours. TGF-β signaling decreases bioluminescence from this reporter.

The BTR FL reporter utilizes luciferase complementation to detect phosphorylation of the sensor by TGF-βR1. Expression of NL is regulated by a promoter with a SMAD binding element (SBE), so canonical TGF-β signaling increases bioluminescence. R values denote goodness of fit for linear regression analysis of data.Ī) Schematic of NL and FL reporter constructs. Graph shows mean values + SEM for each enzyme (n = 4 per condition). D, E) Graphs display bioluminescence quantified in supernatants (D) and intact cells (E) from listed numbers of 231-NL and 231-GL cells. Ratios of band intensities for NL or GL relative to GAPDH were quantified by Image J. Western blots were stripped and probed for GAPDH as a control for equal loading. C) Total cell lysates from 231-NL or 231-GL cells were probed with antibodies to NL or GL, respectively. Inset shows actual photon flux values for the initial 30 seconds of imaging for 231-NL and 231-GL cells. Graph shows mean values ± SEM for bioluminescence relative to initial photon flux for each enzyme measured for over the initial 30 seconds of imaging after adding substrate (n = 4 per condition). B) Equal concentrations of furimazine or coelenterazine were added to MDA-MB-231 human breast cancer cells stably transduced with NL (231-NL) or GL (231-GL), respectively. In some panels, error bars are smaller than corresponding symbols for each data point. Graph displays mean values + SEM for bioluminescence from each luciferase enzyme normalized to co-transfected FL (n = 4 per condition). Our results establish NL as a new reporter for bioluminescence imaging studies in intact cells and living mice that will expand imaging of signal transduction in normal physiology, disease, and drug development.Ī) We measured bioluminescence in supernatants and intact 293T cells transiently transfected with NL or GL. We combined NL and firefly luciferase reporters to quantify two key steps in transforming growth factor β signaling in intact cells and living mice, establishing a novel dual luciferase imaging strategy for quantifying signal transduction and drug targeting. Changes in bioluminescence from NL over time could be used to quantify tumor growth, and secreted NL was detectable in small volumes of serum. We demonstrated that NL could be imaged in superficial and deep tissues in living mice, although the detection of NL in deep tissues was limited by emission of predominantly blue light by this enzyme. To advance dual reporter bioluminescence molecular imaging, we tested a recently developed, adenosine triphosphate–independent luciferase enzyme from Oplophorus gracilirostris (NanoLuc ) as a reporter for animal imaging. A relatively small number of luciferases are available for imaging studies, substantially limiting the ability to image multiple molecular and cellular events, as done commonly with fluorescence imaging. Bioluminescence imaging is widely used for cell-based assays and animal imaging studies in biomedical research and drug development, capitalizing on the high signal to background of this technique.

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