BACKGROUND
forkhead box o  proteins are emerging as transcriptional integrators of pathways that regulate a variety of cellular processes, including differentiation, metabolism, stress response, cell cycle and apoptosis  <cit> .
foxo transcription factors have been proposed to act as bona fide tumor suppressors due to their inhibitory effects on cell cycle and survival  <cit> , properties mediated by their binding as monomers to consensus dna binding sites.
their transcriptional activity is governed by a network of signaling events, the best recognized of which is the phosphorylation of foxo proteins at three highly conserved serine and threonine residues by akt that provokes its association with 14-3- <dig> protein and in turn, the nuclear exclusion of phospho-foxo.
however, the relocation of foxo from the nucleus to the cytoplasm alone cannot account for the inhibitory effect of pi3k/akt signaling on foxo activity since a nuclear form of foxo <dig> in which the nuclear export sequence is disrupted is still inhibited by the pi3k/akt pathway  <cit> .
indeed, the introduction of a negative charge in the positively charged dna binding domain by means of foxo phosphorylation at the second of the three akt consensus sites inhibits dna binding of foxo  <cit> .
the foxo dna interaction is also regulated by the transfer of acetyl groups to lysine residues in foxo proteins by the histone acetyltransferases  cbp and p <dig>  <cit> , which alters the dna binding capacity of foxo <dig> and foxo3a  <cit> .
conversely, sirt <dig> deacetylases deacetylate foxo factors and regulate their dna binding at specific target genes.
taken together, these observations suggest that translocation and transactivation are different and separate means to regulate foxo.
however, large scale tools are not available to assess the different levels of foxo regulation.
therefore systematic chemical genetic or loss of function studies to investigate the complex regulation of foxo factors have been limited only to certain aspects  <cit> .
in anticancer drug discovery, much effort is directed towards identifying small molecule inhibitors of pi3k/akt signaling using cell based high content screening.
in particular, monitoring the intracellular localization of foxo transcription factors has been used to screen large numbers of small molecules  <cit> .
despite being commonly used as a reporter-gene system in drug discovery, luciferase-based transcriptional assays have not been applied to massive compound screens for pi3k/akt inhibitors.
inhibiting the pi3k/akt pathway causes foxo3a to remain in the cell nucleus and subsequently, it induces the transcription of downstream genes.
to take advantage of these regulatory features we generated the stable u2transluc dual assay cell line that expresses foxo responsive luciferase activity and gfp labelled foxo.
thus, u2transluc can be used to simultaneously monitor the intracellular translocation and the transcriptional activity of foxo proteins.
we have used this cell line in an attempt to identify small molecules that interfere with foxo signaling.
RESULTS
generation and testing of luciferase reporter gene constructs
foxo proteins drive the transcription of downstream genes by binding to the ttgtttac foxo responsive enhancer element, generally referred to as a daf- <dig> family protein-binding element   <cit> .
to take advantage of these regulatory features, we engineered several luciferase reporter constructs that contained one to six copies of the dbe consensus cassette in front of a sv <dig> minimal viral promoter that was linked to a luciferase reporter gene.
the resulting reporter gene construct were designated as pgl-1xdbe, pgl-2xdbe, pgl-3xdbe, pgl-4xdbe, pgl-5xdbe and pgl-6xdbe , and the luciferase activity driven by foxo from these constructs was evaluated after they were transiently transfected into u2os osteosarcoma cells.
since endogenous foxo3a is only weakly expressed in these cells, ectopic foxo3a also had to be expressed to achieve acceptable basal levels of luciferase activity .
in transient co-transfection assays, all the luciferase reporter constructs that carried foxo responsive dbe elements produced a significant increase in luciferase activity when compared to the empty pgl3-promoter vector.
constructs that contained three or six copies of the dbe element conferred significantly stronger foxo-dependent transcriptional activity than those with one, two, four or five copies .
a reporter plasmid that carried three copies of a mutated dbe  element did not promote significant luciferase activity, confirming the specificity of the original constructs for foxo-mediated transcriptional activity.
in order to evaluate the responsiveness of the reporter constructs to the inhibition of the pi3k/akt pathway, we transiently co-transfected the pgl-3xdbe or pgl-6xdbe construct with foxo3a into u2os cells and then treated them with the pi3k inhibitor, ly <dig> .
inhibition of the pi3k pathway increased foxo-dependent transcription from both the pgl-3xdbe and pgl-6xdbe constructs approximately 3-fold .
by contrast, activation of the pi3k/akt signaling pathway following exposure to insulin decreased this luciferase activity.
however, the differences between the transcriptional activity of foxo3a in untreated and insulin treated u2os cells were small, indicating that steady-state level of pi3k/akt activity was already quite high.
in addition, to confirm the specificity of our system we examined the effect of constitutive foxo activation by co-expressing the pgl-3xdbe or pgl-6xdbe reporter construct with foxo3a-a <dig>  a constitutively active form of foxo3a in which the three pi3k-dependent phosphorylation sites have been mutated to alanine.
the expression of foxo3a-a <dig> induced a strong increase in pgl-3xdbe or pgl-6xdbe driven luciferase activity.
together, these data indicate that the firefly luciferase-based read-out of foxo activity is very specific, and that it provides a large window to measure any upregulation in the response .
since the responsiveness of the triple tandem repeat of dbe  to pi3k inhibition was slightly higher than that of pgl-6xdbe, the p3xdbe-luc construct was used to generate the stable cell assay line, u2transluc.
generation of the assay cell line, u2transluc
we recently reported the use of a high-throughput cellular imaging assay to monitor the nucleo-cytoplasmic translocation of a gfp-foxo3a fusion protein in u2os cells  for a chemical genetic study  <cit> .
in order to generate an assay system that combines the benefits of large-scale image-based analysis and luciferase-based end point measurements, we examined the compatibility of transcriptional and translocational reporters expressed in a single cell line.
we first explored whether the gfp-tagged foxo protein is still capable of driving luciferase expression from foxo responsive reporters.
we compared the reporter activity of the pgl-3xdbe construct or the mutated version  co-expressed with either untagged foxo3a wt protein or egfp-tagged foxo3a.
hence, we transiently transfected untagged foxo3a and egfp-foxo3a into u2os cells and measured the luciferase activities  <dig> hours after exposure to ly <dig> or dmso.
the n-terminal egfp tag did not significantly affect transcriptional function of foxo3a in u2os cells  indicating the compatibility of luminescent and fluorescent read-outs in a single cell line.
in addition, we compared the level of luciferase activity driven by the pgl-3xdbe construct in the context of stable egfp-foxo3a expression in u2foxreloc cells  <cit>  and with the transient co-expression of the fluorescent fusion protein.
the luciferase expression in the presence of stable egfp-foxo3a expression was only slightly lower than that in u2os cells transiently overexpressing egfp-foxo3a.
to stably integrate the pgl-3xdbe construct into the genome of u2foxreloc cells, we modified the pgl3-promoter construct by inserting a puromycin-resistance cassette to obtain pglpuro-3xdbe.
puromycin-resistant stable clones were generated  and one was chosen for further use based on foxo-induced luciferase activity.
the use of gfp fusion protein for normalization
we examined whether the expression of the gfp fusion protein could be used to normalize the transcriptional reporter gene to the changes in cell viability.
u2transluc cells were seeded and allowed to attach to the wells overnight.
subsequently, they were incubated with increasing doses of sodium azide, a known inhibitor of the respiratory chain that provokes necrosis in many cell types  <cit> .
cell viability was estimated through a crystal violet assay or through the intensity of the fluorescence measured in each well.
both methods take advantage of the fact that when epithelial cells undergo apoptosis or necrosis, they detach from plates and are no longer detectable by these assays.
after a  <dig> hour exposure to  <dig> % sodium azide the viability of u2transluc cells was slightly reduced with no significant differences between crystal violet or fluorescent intensity read-outs.
higher doses of sodium azide dramatically compromised u2transluc viability, as reflected by both measures of viability.
a normalization procedure using crystal violet staining or the gfp-foxo mediated fluorescent signal resulted in similar indices of viability , indicating that the fluorescent reporter protein can be used to monitor the viability of u2transluc cells.
multiplexed u2transluc assay
the u2transluc-based assay was formatted for 96-well plates and the workflow has been automated.
to assess the specificity of the transcriptional and the translocational read-out, we challenged the u2transluc system with small molecule inhibitors under living cell conditions in order to observe the translocation of the fluorescent reporter protein and luciferase activity as the transcriptional end point.
we exposed u2transluc cells to a panel of test compounds with known biological activity.
three different doses were used that resulted in a range of final concentrations greater than two orders of magnitude around the ic <dig> value for each individual compound.
the live cells were stained with the fluorescent nuclear dye draq <dig> and images were obtained on an automatic microscope.
the nuclear/cytoplasmic  fluorescence intensity ratios were determined and the percentage of cells per well displaying nuclear translocation or the inhibition of nuclear export was calculated.
compounds that induced the nuclear accumulation of the fluorescent signal above 60% of that obtained from wells treated with ly <dig> were considered as hits.
several compounds known to inhibit pi3k activity or the nuclear export machinery fulfilled these criteria .
as expected from the results of a previous study in u2foxreloc cells  <cit> , the pi3k inhibitors pi- <dig> and ly <dig> were capable of inducing foxo translocation into the nucleus of u2transluc cells.
by contrast, known activators of the pi3k/akt pathway produced little nuclear localization of gfp-foxo, including egf, igf and pdgf.
the exposure of u2transluc cells to the nuclear export inhibitors leptomycin b or ratjadone a provoked the nuclear accumulation of fluorescent signal, as shown previously for u2foxreloc cells  <cit> .
conversely, through the measurement of the foxo-dependent production of firefly luciferase, leptomycin b and ratjadone a exerted no significant effect on the transcriptional activity of foxo .
finally, exposure to pi- <dig> and ly <dig> triggered a dramatic increase in foxo-dependent luciferase activity.
together, these data show that the u2transluc assay serves as an automated high throughput assay that can identify inhibitors of pi3k/akt signaling through the high content analysis of protein translocation in conjunction with an independent analysis of the transcriptional activity of foxo transcriptions factors.
discussion and
CONCLUSIONS
foxo transcription factors are tightly regulated at different levels, including their intracellular translocation and transcriptional activity  <cit> .
here we report the generation of a multiplexed assay system that is capable of simultaneously monitoring these signaling events.
the assay system is based on u2transluc cells that stably express two different reporter constructs.
the intracellular translocation of foxo factors is followed using a fluorescent tagged foxo reporter protein, whereas transcriptional activity is monitored via foxo-dependent luciferase production.
we show here that the fluorescent and luminescent read-outs are compatible in the same cell.
the design of the u2transluc screen presented here offers some major advantages over more classical cellular assays.
primary and secondary screens of small molecule compounds are usually performed in different cell systems.
by contrast, the u2transluc provides the possibility of using two different read-outs within the same experiment, enabling a direct comparison of the hits from each of these.
these hits might be divided into translocation, transactivation and dual hits, and they may in turn be analyzed according to a corresponding hit ranking.
the u2transluc assay does not need the introduction of additional plasmids and hence, it avoids the limitations associated with transfection procedures.
the use of the gfp-foxo fusion protein provides a multifunctional tool that allows for versatile assay read outs, measurement of cell viability/number and sorting and maintaining a homogeneous cell assay population.
we show that the fluorescent signal from the gfp-foxo fusion protein is suitable to monitor viability and it can be used to normalize the values obtained by measuring firefly luciferase activity.
furthermore, the fluorescent foxo functions as a reporter protein for foxo translocation and as an effector protein that drives dbe-dependent luiferase production.
on the other hand the gfp-foxo fusion protein offers the possibility to control the level of expression of the transcriptional effector in the luciferase assay, and at the same time it serves as a sorting tool to maintain a homogenous cell population.
thus, the multifunctional use of gfp-foxo allows also to reduce the variability of the assay.
the u2transluc is an image based high throughput assay that enables compounds that produce artifacts and cytotoxicity to be identified on a single cell basis.
finally, the u2transluc assay design might be adaptable to any transcription factor that undergoes nucleo-cytoplasmic shuttling.
we challenged the u2transluc system with chemical agents with known biological activity and were able to identify known pi3k inhibitors as double-hits that score in the translocation and the transactivation read-out.
by contrast, chemical probes that inhibit the general nuclear export machinery failed to produce an increase in luciferase activity although gfp-foxo was trapped in the cell nucleus.
in summary, our data demonstrate that u2transluc is a sensitive and robust assay to identify small-molecule inhibitors of signaling events that regulate the subcellular localization and/or the transcriptional activity of foxo proteins.
the signaling events identified here include pi3k/akt signaling and nuclear export.
our data raise the expectation that a more extensive chemical interrogation of the u2transluc read-outs could lead to the identification of new molecular targets and small molecules that might contribute to the development of more potent therapeutic agents to treat tumors.

 forkhead box o proteins are emerging as transcriptional integrators of pathways that regulate a variety of cellular processes, including differentiation, metabolism, stress response, cell cycle and apoptosis. foxo transcription factors have been proposed to act as bona fide tumor suppressors due to their inhibitory effects on cell cycle and survival, properties mediated by their binding as monomers to consensus dna binding sites. their transcriptional activity is governed by a network of signaling events, the best recognized of which is the phosphorylation of foxo proteins at three highly conserved serine and threonine residues by akt that provokes its association with 14-3- protein and in turn, the nuclear exclusion of phospho-foxo. however, the relocation of foxo from the nucleus to the cytoplasm alone cannot account for the inhibitory effect of pi3k/akt signaling on foxo activity since a nuclear form of foxo in which the nuclear export sequence is disrupted is still inhibited by the pi3k/akt pathway. indeed, the introduction of a negative charge in the positively charged dna binding domain by means of foxo phosphorylation at the second of the three akt consensus sites inhibits dna binding of foxo. the foxo dna interaction is also regulated by the transfer of acetyl groups to lysine residues in foxo proteins by the histone acetyltransferases cbp and p, which alters the dna binding capacity of foxo and foxo3a. conversely, sirt deacetylases deacetylate foxo factors and regulate their dna binding at specific target genes. taken together, these observations suggest that translocation and transactivation are different and separate means to regulate foxo. however, large scale tools are not available to assess the different levels of foxo regulation. therefore systematic chemical genetic or loss of function studies to investigate the complex regulation of foxo factors have been limited only to certain aspects. in anticancer drug discovery, much effort is directed towards identifying small molecule inhibitors of pi3k/akt signaling using cell based high content screening. in particular, monitoring the intracellular localization of foxo transcription factors has been used to screen large numbers of small molecules. despite being commonly used as a reporter-gene system in drug discovery, luciferase-based transcriptional assays have not been applied to massive compound screens for pi3k/akt inhibitors. inhibiting the pi3k/akt pathway causes foxo3a to remain in the cell nucleus and subsequently, it induces the transcription of downstream genes. to take advantage of these regulatory features we generated the stable u2transluc dual assay cell line that expresses foxo responsive luciferase activity and gfp labelled foxo. thus, u2transluc can be used to simultaneously monitor the intracellular translocation and the transcriptional activity of foxo proteins. we have used this cell line in an attempt to identify small molecules that interfere with foxo signaling.  generation and testing of luciferase reporter gene constructs foxo proteins drive the transcription of downstream genes by binding to the ttgtttac foxo responsive enhancer element, generally referred to as a daf- family protein-binding element. to take advantage of these regulatory features, we engineered several luciferase reporter constructs that contained one to six copies of the dbe consensus cassette in front of a sv minimal viral promoter that was linked to a luciferase reporter gene. the resulting reporter gene construct were designated as pgl-1xdbe, pgl-2xdbe, pgl-3xdbe, pgl-4xdbe, pgl-5xdbe and pgl-6xdbe, and the luciferase activity driven by foxo from these constructs was evaluated after they were transiently transfected into u2os osteosarcoma cells. since endogenous foxo3a is only weakly expressed in these cells, ectopic foxo3a also had to be expressed to achieve acceptable basal levels of luciferase activity. in transient co-transfection assays, all the luciferase reporter constructs that carried foxo responsive dbe elements produced a significant increase in luciferase activity when compared to the empty pgl3-promoter vector. constructs that contained three or six copies of the dbe element conferred significantly stronger foxo-dependent transcriptional activity than those with one, two, four or five copies. a reporter plasmid that carried three copies of a mutated dbe element did not promote significant luciferase activity, confirming the specificity of the original constructs for foxo-mediated transcriptional activity. in order to evaluate the responsiveness of the reporter constructs to the inhibition of the pi3k/akt pathway, we transiently co-transfected the pgl-3xdbe or pgl-6xdbe construct with foxo3a into u2os cells and then treated them with the pi3k inhibitor, ly. inhibition of the pi3k pathway increased foxo-dependent transcription from both the pgl-3xdbe and pgl-6xdbe constructs approximately 3-fold. by contrast, activation of the pi3k/akt signaling pathway following exposure to insulin decreased this luciferase activity. however, the differences between the transcriptional activity of foxo3a in untreated and insulin treated u2os cells were small, indicating that steady-state level of pi3k/akt activity was already quite high. in addition, to confirm the specificity of our system we examined the effect of constitutive foxo activation by co-expressing the pgl-3xdbe or pgl-6xdbe reporter construct with foxo3a-a a constitutively active form of foxo3a in which the three pi3k-dependent phosphorylation sites have been mutated to alanine. the expression of foxo3a-a induced a strong increase in pgl-3xdbe or pgl-6xdbe driven luciferase activity. together, these data indicate that the firefly luciferase-based read-out of foxo activity is very specific, and that it provides a large window to measure any upregulation in the response. since the responsiveness of the triple tandem repeat of dbe to pi3k inhibition was slightly higher than that of pgl-6xdbe, the p3xdbe-luc construct was used to generate the stable cell assay line, u2transluc. generation of the assay cell line, u2transluc we recently reported the use of a high-throughput cellular imaging assay to monitor the nucleo-cytoplasmic translocation of a gfp-foxo3a fusion protein in u2os cells for a chemical genetic study. in order to generate an assay system that combines the benefits of large-scale image-based analysis and luciferase-based end point measurements, we examined the compatibility of transcriptional and translocational reporters expressed in a single cell line. we first explored whether the gfp-tagged foxo protein is still capable of driving luciferase expression from foxo responsive reporters. we compared the reporter activity of the pgl-3xdbe construct or the mutated version co-expressed with either untagged foxo3a wt protein or egfp-tagged foxo3a. hence, we transiently transfected untagged foxo3a and egfp-foxo3a into u2os cells and measured the luciferase activities hours after exposure to ly or dmso. the n-terminal egfp tag did not significantly affect transcriptional function of foxo3a in u2os cells indicating the compatibility of luminescent and fluorescent read-outs in a single cell line. in addition, we compared the level of luciferase activity driven by the pgl-3xdbe construct in the context of stable egfp-foxo3a expression in u2foxreloc cells and with the transient co-expression of the fluorescent fusion protein. the luciferase expression in the presence of stable egfp-foxo3a expression was only slightly lower than that in u2os cells transiently overexpressing egfp-foxo3a. to stably integrate the pgl-3xdbe construct into the genome of u2foxreloc cells, we modified the pgl3-promoter construct by inserting a puromycin-resistance cassette to obtain pglpuro-3xdbe. puromycin-resistant stable clones were generated and one was chosen for further use based on foxo-induced luciferase activity. the use of gfp fusion protein for normalization we examined whether the expression of the gfp fusion protein could be used to normalize the transcriptional reporter gene to the changes in cell viability. u2transluc cells were seeded and allowed to attach to the wells overnight. subsequently, they were incubated with increasing doses of sodium azide, a known inhibitor of the respiratory chain that provokes necrosis in many cell types. cell viability was estimated through a crystal violet assay or through the intensity of the fluorescence measured in each well. both methods take advantage of the fact that when epithelial cells undergo apoptosis or necrosis, they detach from plates and are no longer detectable by these assays. after a hour exposure to % sodium azide the viability of u2transluc cells was slightly reduced with no significant differences between crystal violet or fluorescent intensity read-outs. higher doses of sodium azide dramatically compromised u2transluc viability, as reflected by both measures of viability. a normalization procedure using crystal violet staining or the gfp-foxo mediated fluorescent signal resulted in similar indices of viability, indicating that the fluorescent reporter protein can be used to monitor the viability of u2transluc cells. multiplexed u2transluc assay the u2transluc-based assay was formatted for 96-well plates and the workflow has been automated. to assess the specificity of the transcriptional and the translocational read-out, we challenged the u2transluc system with small molecule inhibitors under living cell conditions in order to observe the translocation of the fluorescent reporter protein and luciferase activity as the transcriptional end point. we exposed u2transluc cells to a panel of test compounds with known biological activity. three different doses were used that resulted in a range of final concentrations greater than two orders of magnitude around the ic value for each individual compound. the live cells were stained with the fluorescent nuclear dye draq and images were obtained on an automatic microscope. the nuclear/cytoplasmic fluorescence intensity ratios were determined and the percentage of cells per well displaying nuclear translocation or the inhibition of nuclear export was calculated. compounds that induced the nuclear accumulation of the fluorescent signal above 60% of that obtained from wells treated with ly were considered as hits. several compounds known to inhibit pi3k activity or the nuclear export machinery fulfilled these criteria. as expected from the results of a previous study in u2foxreloc cells, the pi3k inhibitors pi- and ly were capable of inducing foxo translocation into the nucleus of u2transluc cells. by contrast, known activators of the pi3k/akt pathway produced little nuclear localization of gfp-foxo, including egf, igf and pdgf. the exposure of u2transluc cells to the nuclear export inhibitors leptomycin b or ratjadone a provoked the nuclear accumulation of fluorescent signal, as shown previously for u2foxreloc cells. conversely, through the measurement of the foxo-dependent production of firefly luciferase, leptomycin b and ratjadone a exerted no significant effect on the transcriptional activity of foxo. finally, exposure to pi- and ly triggered a dramatic increase in foxo-dependent luciferase activity. together, these data show that the u2transluc assay serves as an automated high throughput assay that can identify inhibitors of pi3k/akt signaling through the high content analysis of protein translocation in conjunction with an independent analysis of the transcriptional activity of foxo transcriptions factors. discussion and  foxo transcription factors are tightly regulated at different levels, including their intracellular translocation and transcriptional activity. here we report the generation of a multiplexed assay system that is capable of simultaneously monitoring these signaling events. the assay system is based on u2transluc cells that stably express two different reporter constructs. the intracellular translocation of foxo factors is followed using a fluorescent tagged foxo reporter protein, whereas transcriptional activity is monitored via foxo-dependent luciferase production. we show here that the fluorescent and luminescent read-outs are compatible in the same cell. the design of the u2transluc screen presented here offers some major advantages over more classical cellular assays. primary and secondary screens of small molecule compounds are usually performed in different cell systems. by contrast, the u2transluc provides the possibility of using two different read-outs within the same experiment, enabling a direct comparison of the hits from each of these. these hits might be divided into translocation, transactivation and dual hits, and they may in turn be analyzed according to a corresponding hit ranking. the u2transluc assay does not need the introduction of additional plasmids and hence, it avoids the limitations associated with transfection procedures. the use of the gfp-foxo fusion protein provides a multifunctional tool that allows for versatile assay read outs, measurement of cell viability/number and sorting and maintaining a homogeneous cell assay population. we show that the fluorescent signal from the gfp-foxo fusion protein is suitable to monitor viability and it can be used to normalize the values obtained by measuring firefly luciferase activity. furthermore, the fluorescent foxo functions as a reporter protein for foxo translocation and as an effector protein that drives dbe-dependent luiferase production. on the other hand the gfp-foxo fusion protein offers the possibility to control the level of expression of the transcriptional effector in the luciferase assay, and at the same time it serves as a sorting tool to maintain a homogenous cell population. thus, the multifunctional use of gfp-foxo allows also to reduce the variability of the assay. the u2transluc is an image based high throughput assay that enables compounds that produce artifacts and cytotoxicity to be identified on a single cell basis. finally, the u2transluc assay design might be adaptable to any transcription factor that undergoes nucleo-cytoplasmic shuttling. we challenged the u2transluc system with chemical agents with known biological activity and were able to identify known pi3k inhibitors as double-hits that score in the translocation and the transactivation read-out. by contrast, chemical probes that inhibit the general nuclear export machinery failed to produce an increase in luciferase activity although gfp-foxo was trapped in the cell nucleus. in summary, our data demonstrate that u2transluc is a sensitive and robust assay to identify small-molecule inhibitors of signaling events that regulate the subcellular localization and/or the transcriptional activity of foxo proteins. the signaling events identified here include pi3k/akt signaling and nuclear export. our data raise the expectation that a more extensive chemical interrogation of the u2transluc read-outs could lead to the identification of new molecular targets and small molecules that might contribute to the development of more potent therapeutic agents to treat tumors. 
u2transluc is suitable for high throughput screening and can identify small molecules that interfere with foxo signaling at different levels. the u2transluc system is based on a stable cell line expressing a gfp-tagged foxo transcription factor and a luciferase reporter gene under the control of human foxo-responsive enhancers. combining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. we developed u2transluc, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method. the u2transluc assay measures nuclear-cytoplasmic foxo shuttling and foxo-driven transcription, providing a means to analyze these two key features of foxo regulation in the same experiment. we challenged the u2transluc system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity. independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. the u2transluc assay facilitates the maintenance and monitoring of homogeneous foxo transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability.
 independent luciferase reporter assays and fluorescent translocation assays have been successfully used in drug discovery for several molecular targets. we developed u2transluc, an assay system in which luciferase and fluorescent read-outs can be multiplexed to provide a powerful cell-based high content screening method.  the u2transluc system is based on a stable cell line expressing a gfp-tagged foxo transcription factor and a luciferase reporter gene under the control of human foxo-responsive enhancers. the u2transluc assay measures nuclear-cytoplasmic foxo shuttling and foxo-driven transcription, providing a means to analyze these two key features of foxo regulation in the same experiment. we challenged the u2transluc system with chemical probes with known biological activities and we were able to identify compounds with translocation and/or transactivation capacity.  combining different biological read-outs in a single cell line offers significant advantages over conventional cell-based assays. the u2transluc assay facilitates the maintenance and monitoring of homogeneous foxo transcription factor expression and allows the reporter gene activity measured to be normalized with respect to cell viability. u2transluc is suitable for high throughput screening and can identify small molecules that interfere with foxo signaling at different levels. 