Dual Reporter System Research Articles

Distinct Enhancer Activity of Oct4 in Naive and Primed Mouse Pluripotency

SummaryNaive and primed pluripotent stem cells (PSCs) and germ cells express the Oct4 gene. The Oct4 gene contains two cis-regulatory elements, the distal enhancer (DE) and proximal enhancer (PE), which differentially control Oct4 expression in a cell-type-specific and stage-specific manner. Here, we generated double transgenic mice carrying both Oct4-ΔPE-GFP and Oct4-ΔDE-tdTomato (RFP), enabling us to simultaneously monitor the activity of DE and PE. Oct4 expression is stage-specifically regulated by DE and PE during embryonic and germ cell development. Using this dual reporter system, we successfully cultured pure populations of naive (GFP+RFP−) and primed (GFP−RFP+) PSCs. We found that GFP+RFP− cells were metastable (not naive) in serum-containing medium; stable naive pluripotent cells were observed in medium containing two inhibitors (Meki and GSKi) but lacked serum. Finally, we suggest that the activity of Oct4 DE and PE is regulated by the repressive histone marks and DNA methylation in a cell-type-specific manner.

LadS is a calcium-responsive kinase that induces acute-to-chronic virulence switch in Pseudomonas aeruginosa.

Virulence of pathogenic bacteria is a tightly controlled process to facilitate invasion and survival in host tissues. Although pathways controlling virulence have been defined in detail, signals modulating these processes are poorly understood. The opportunistic pathogen Pseudomonas aeruginosa causes acute and chronic infections in humans. Disease progression is typically associated with a loss of acute virulence and the emergence of biofilms and chronic behaviour. The acute-to-chronic switch is governed by the global Gac/Rsm pathway. Using a newly developed acute-chronic dual reporter system we show that calcium stimulates the Gac/Rsm pathway via the Gac-associated hybrid histidine kinase LadS. We show that calcium binds to the periplasmic DISMED2 sensor domain of LadS to activate its kinase activity. Activation of the Gac/Rsm pathway by calcium leads to a switch to the chronic program and confers drug tolerance by reducing P. aeruginosa growth rate. Clinical isolates from cystic fibrosis airways retain their calcium response during chronic infections. Our data imply that calcium sensing evolved as an adaptation to the opportunistic lifestyle of P. aeruginosa and that calcium serves as a host signal to balance acute-to-chronic behaviour during infections. Establishing calcium signalling in host-pathogen interaction adds to growing evidence indicating key roles for calcium in bacterial signalling.

MiR-206 enhances radiosensitivity of glioma cells by targeting MAPK1 pathway

Objective To investigate the signaling pathway probably targeted by miR-206 in regulation of the radiosensitivity of glioma cells, and to provide a basis for further understanding of its regulatory mechanism. Methods The miR-206 mimic or miR-206 inhibitor was transfected into U87 glioma cells. The activity of the MAPK pathway was inhibited by PD98059. The cells were exposed to radiation. MTT assay and colony formation assay were used to determine the changes in the radiosensitivity of cells. Quantitative RT-PCR and Western blot were used to determine the expression of miR-206 and MAPK1, respectively. TargetScan prediction and dual luciferase reporter system were used to verify the interaction between miR-206 and MAPK1. Results After exposure to radiation, the glioma cells had downregulated expression of miR-206 and upregulated expression of MAPK1. Overexpression of miR-206 induced by the miR-206 mimic reduced cell proliferation and colony formation ability and enhanced the radiosensitivity; inhibition of miR-206 expression by the miR-206 inhibitor reversed the effects of the miR-206 mimic. MAPK1 was predicted to be the possible target gene of miR-206 by TargetScan software. The dual luciferase reporter assay further confirmed the interaction between miR-206 and MAPK1. The expression of MAPK1 was negatively correlated with that of miR-206. The radiosensitivity of glioma cells was enhanced when the MAPK pathway was blocked by the inhibitor. Conclusions miR-206 probably targets MAPK1. It may regulate the radiosensitivity of glioma cells by inhibiting the activity of the MAPK signaling pathway. Key words: miR-206 gene; MAPK1 pathway; U87 cell line; Radiosensitivity

The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states.

BackgroundThe epithelial to mesenchymal transition (EMT) has been implicated in metastasis and therapy resistance of carcinomas and can endow cancer cells with cancer stem cell (CSC) properties. The ability to detect cancer cells that are undergoing or have completed EMT has typically relied on the expression of cell surface antigens that correlate with an EMT/CSC phenotype. Alternatively these cells may be permanently marked through Cre-mediated recombination or through immunostaining of fixed cells. The EMT process is dynamic, and these existing methods cannot reveal such changes within live cells. The development of fluorescent sensors that mirror the dynamic EMT state by following the expression of bona fide EMT regulators in live cells would provide a valuable new tool for characterizing EMT. In addition, these sensors will allow direct observation of cellular plasticity with respect to the epithelial/mesenchymal state to enable more effective studies of EMT in cancer and development.ResultsWe generated a lentiviral-based, dual fluorescent reporter system, designated as the Z-cad dual sensor, comprising destabilized green fluorescent protein containing the ZEB1 3′ UTR and red fluorescent protein driven by the E-cadherin (CDH1) promoter. Using this sensor, we robustly detected EMT and mesenchymal to epithelial transition (MET) in breast cancer cells by flow cytometry and fluorescence microscopy. Importantly, we observed dynamic changes in cellular populations undergoing MET. Additionally, we used the Z-cad sensor to identify and isolate minor subpopulations of cells displaying mesenchymal properties within a population comprising predominately epithelial-like cells. The Z-cad dual sensor identified cells with CSC-like properties more effectively than either the ZEB1 3′ UTR or E-cadherin sensor alone.ConclusionsThe Z-cad dual sensor effectively reports the activities of two factors critical in determining the epithelial/mesenchymal state of carcinoma cells. The ability of this stably integrating dual sensor system to detect dynamic fluctuations between these two states through live cell imaging offers a significant improvement over existing methods and helps facilitate the study of EMT/MET plasticity in response to different stimuli and in cancer pathogenesis. Finally, the versatile Z-cad sensor can be adapted to a variety of in vitro or in vivo systems to elucidate whether EMT/MET contributes to normal and disease phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0269-y) contains supplementary material, which is available to authorized users.

A Bacteriophage-Acquired O-Antigen Polymerase (Wzyβ) from P. aeruginosa Serotype O16 Performs a Varied Mechanism Compared to Its Cognate Wzyα.

Pseudomonas aeruginosa is a Gram-negative bacterium that produces highly varied lipopolysaccharide (LPS) structures. The O antigen (O-Ag) in the LPS is synthesized through the Wzx/Wzy-dependent pathway where lipid-linked O-Ag repeats are polymerized by Wzy. Horizontal-gene transfer has been associated with O-Ag diversity. The O-Ag present on the surface of serotypes O5 and O16, differ in the intra-molecular bonds, α and β, respectively; the latter arose from the action of three genes in a serotype converting unit acquired from bacteriophage D3, including a β-polymerase (Wzyβ). To further our understanding of O-polymerases, the inner membrane (IM) topology of Wzyβ was determined using a dual phoA-lacZα reporter system wherein random 3′ gene truncations were localized to specific loci with respect to the IM by normalized reporter activities as determined through the ratio of alkaline phosphatase activity to β-galactosidase activity. The topology of Wzyβ developed through this approach was shown to contain two predominant periplasmic loops, PL3 (containing an RX10G motif) and PL4 (having an O-Ag ligase superfamily motif), associated with inverting glycosyltransferase reaction. Through site-directed mutagenesis and complementation assays, residues Arg254, Arg270, Arg272, and His300 were found to be essential for Wzyβ function. Additionally, like-charge substitutions, R254K and R270K, could not complement the wzyβ knockout, highlighting the essential guanidium side group of Arg residues. The O-Ag ligase domain is conserved among heterologous Wzy proteins that produce β-linked O-Ag repeat units. Taking advantage of the recently obtained whole-genome sequence of serotype O16 a candidate promoter was identified. Wzyβ under its native promoter was integrated in the PAO1 genome, which resulted in simultaneous production of α- and β-linked O-Ag. These observations established that members of Wzy-like family consistently exhibit a dual-periplasmic loops topology, and identifies motifs that are plausible to be involved in enzymatic activities. Based on these results, the phage-derived Wzyβ utilizes a different reaction mechanism in the P. aeruginosa host to avoid self-inhibition during serotype conversion.

Elucidating population-wide mycobacterial replication dynamics at the single-cell level.

Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits two fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescence single-cell analysis to characterize intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 h of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by d-cycloserine treatment); this population is likely to be highly enriched for persisters, based on its drug-tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescence reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.

Feedback Regulation and Its Efficiency in Biochemical Networks

Intracellular biochemical networks fluctuate dynamically due to various internal and external sources of fluctuation. Dissecting the fluctuation into biologically relevant components is important for understanding how a cell controls and harnesses noise and how information is transferred over apparently noisy intracellular networks. While substantial theoretical and experimental advancement on the decomposition of fluctuation was achieved for feedforward networks without any loop, we still lack a theoretical basis that can consistently extend such advancement to feedback networks. The main obstacle that hampers is the circulative propagation of fluctuation by feedback loops. In order to define the relevant quantity for the impact of feedback loops for fluctuation, disentanglement of the causally interlocked influence between the components is required. In addition, we also lack an approach that enables us to infer non-perturbatively the influence of the feedback to fluctuation as the dual reporter system does in the feedforward network. In this work, we resolve these problems by extending the work on the fluctuation decomposition and the dual reporter system. For a single-loop feedback network with two components, we define feedback loop gain as the feedback efficiency that is consistent with the fluctuation decomposition for feedforward networks. Then, we clarify the relation of the feedback efficiency with the fluctuation propagation in an open-looped FF network. Finally, by extending the dual reporter system, we propose a conjugate feedback and feedforward system for estimating the feedback efficiency only from the statistics of the system non-perturbatively.

A Dual Receptor and Reporter for Multi-Modal Cell Surface Engineering.

The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction.

Orthogonal control of expression mean and variance by epigenetic features at different genomic loci.

While gene expression noise has been shown to drive dramatic phenotypic variations, the molecular basis for this variability in mammalian systems is not well understood. Gene expression has been shown to be regulated by promoter architecture and the associated chromatin environment. However, the exact contribution of these two factors in regulating expression noise has not been explored. Using a dual-reporter lentiviral model system, we deconvolved the influence of the promoter sequence to systematically study the contribution of the chromatin environment at different genomic locations in regulating expression noise. By integrating a large-scale analysis to quantify mRNA levels by smFISH and protein levels by flow cytometry in single cells, we found that mean expression and noise are uncorrelated across genomic locations. Furthermore, we showed that this independence could be explained by the orthogonal control of mean expression by the transcript burst size and noise by the burst frequency. Finally, we showed that genomic locations displaying higher expression noise are associated with more repressed chromatin, thereby indicating the contribution of the chromatin environment in regulating expression noise.

A platform for rapid generation of single and multiplexed reporters in human iPSC lines.

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript, we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore, we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages, further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays.

Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors.

Most cancer therapies involve a component of treatment that inflicts DNA damage in tumor cells, such as double-strand breaks (DSBs), which are considered the most serious threat to genomic integrity. Complex systems have evolved to repair these lesions, and successful DSB repair is essential for tumor cell survival after exposure to ionizing radiation (IR) and other DNA-damaging agents. As such, inhibition of DNA repair is a potentially efficacious strategy for chemo- and radiosensitization. Homologous recombination (HR) and nonhomologous end-joining (NHEJ) represent the two major pathways by which DSBs are repaired in mammalian cells. Here, we report the design and execution of a high-throughput, cell-based small molecule screen for novel DSB repair inhibitors. We miniaturized our recently developed dual NHEJ and HR reporter system into a 384-well plate-based format and interrogated a diverse library of 20,000 compounds for molecules that selectively modulate NHEJ and HR repair in tumor cells. We identified a collection of novel hits that potently inhibit DSB repair, and we have validated their functional activity in a comprehensive panel of orthogonal secondary assays. A selection of these inhibitors was found to radiosensitize cancer cell lines in vitro, which suggests that they may be useful as novel chemo- and radio sensitizers. Surprisingly, we identified several FDA-approved drugs, including the calcium channel blocker mibefradil dihydrochloride, that demonstrated activity as DSB repair inhibitors and radiosensitizers. These findings suggest the possibility for repurposing them as tumor cell radiosensitizers in the future. Accordingly, we recently initiated a phase I clinical trial testing mibefradil as a glioma radiosensitizer.

Inside Cover: A Dual Reporter System for Detecting RNA Interactions in Bacterial Cells (ChemBioChem 18/2014)

Inside Cover: A Dual Reporter System for Detecting RNA Interactions in Bacterial Cells (ChemBioChem 18/2014)

Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location.

Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes.

A dual reporter system for detecting RNA interactions in bacterial cells.

Detecting RNA-partner interactions in cells is often difficult due to a lack of suitable tools. Here we describe a dual reporter system capable of detecting intracellular interactions in which one of the partners is an RNA. The system utilizes two fluorescent proteins with similar maturation rates but distinct spectral properties, specifically cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). By placing the CFP gene upstream and the YFP gene downstream of an RNA gene of interest, the production of YFP becomes sensitive to RNA-partner interaction, whereas the synthesis of CFP is not disturbed. Therefore, the RNA-partner interaction can be simply measured by the change in the ratio of fluorescence of YFP over CFP. The utility of our approach is demonstrated through verification of three known RNA-partner interactions in the model bacterium Escherichia coli. Our two-reporter strategy should be broadly useful to the study of RNA-targeted interactions in bacteria.

Vaginal challenge with an SIV-based dual reporter system reveals that infection can occur throughout the upper and lower female reproductive tract.

The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection.

Construction and validation of a dual-luciferase reporter gene system for screening and evaluating anti-liver fibrosis drugs that inhibit transcription of the gene encoding collagen I, chain a1

To construct a dual-reporter gene system that will be applicable for use as a tool to screen and evaluate therapeutic drug compounds that inhibit transcription of the gene encoding collagen I, chain at1 (COL1A1). The full-length eDNA of transforming growth factor beta1 (TGFbeta1) was cloned by RT-PCR and inserted into two vectors, pcDNA3.1 and pJW4303, for construction of two eukaryotic expression vectors, pcDNA3.1-TGFbeta1 and pJW4303-TGFbeta1.Next, the promoter region of COL1A1, cloned by PCR using human genome DNA as template, was inserted into the vector pGL4.29 to construct the reporter gene vector, pGL4.29-COL1A1 promoter.All three recombinant vectors were verified by restriction enzyme digestion and DNA sequencing.Either the pcDNA3.1-TGFbeta1 or pJW4303-TGFbeta1 vector along with the pGL4.29-COL1A1 promoter vector or a pRL-null, control reporter, vector were co-transfected into the LX-2 human hepatic stellate cells to establish the transcription-activated dualreporter gene system.This system was used as a cell model for screening anti-liver fibrosis compounds that inhibit the transcription of COL1A1.Dexamethasone, a model drug that is known to inhibit the expression of COL1A1, was used as a control to validate the dual-reporter gene system. The two TGFbeta1-expressing vectors and the reporter gene vector containing the promoter region of COL1A1 were successfully constructed.The results of a dual-reporter gene assay showed that TGFbeta1 co-expression increased the activity of the COL1A1 promoter by above 200-fold (t =21.78, P =0.0001), whereas in the absence of TGF31 co-expression the activity was below 2-fold (t =3.396, P =0.0274).The transcriptionactivated dual-reporter gene system was successfully established.The model drug, dexamethasone, effectively inhibited the activity of the COL 1A1 promoter in dose-dependent manner; the activity decreased 29.6% with 10 mumol/L dexamethasone (t =4.140, P =0.0144) and 53.9% with 100 mumol/L (t =6.193, P =0.0035). The dual-luciferase reporter system of TGFbeta1 and COL1A1 co-expression developed here can be used as a cell model to screen and evaluate anti-liver fibrosis compounds that inhibit activity of the COL1A1.

A dual-reporter, diagnostic vector for prostate cancer detection and tumor imaging.

Detection of prostate-specific antigen (PSA) as a screening strategy for prostate cancer is limited by the inability of the PSA test to differentiate between malignant cancer and benign hyperplasia. Here, we report the use of a cancer-specific promoter, inhibition of differentiation-1 (Id1), to drive a dual-reporter system (Ad5/3-Id1-SEAP-Id1-mCherry) designed for detection of prostate cancer using a blood-based reporter-secreted embryonic alkaline phosphatase (SEAP) and tumor visualization using a fluorescent reporter protein, mCherry. In human prostate tumors, Id1 levels are correlated with increased Gleason grade and disease progression. To evaluate the performance of the dual-reporter system, a prostate cell panel with varying aggressive phenotypes was tested. Following infection with the Ad5/3-Id1-SEAP-Id1-mCherry vector, expression of the SEAP and mCherry reporters was shown to increase with increasing levels of cellular Id1. No correlation was observed between Id1 and PSA. To evaluate in vivo performance, flank tumors were grown in athymic male mice using three prostate cancer cell lines. Following intra-tumoral injection of the vector, tumors formed by cells with high Id1 had the greatest reporter expression. Interestingly, tumors with the lowest levels of Id1 and reporter expression produced the greatest amounts of PSA. These data support the use of Ad5/3-Id1-SEAP-Id1-mCherry as a predictor of prostate cancer malignancy and as a strategy for tumor localization.

PDsRed-EGFPmtag-, an effective dual fluorescent reporter system for cell-based screens of premature termination codon.

A large number of inherited diseases are caused by premature termination codon (PTC) mutations that lead to the degradation of mRNA template. In this report, we developed a dual fluorescent reporter that relied the feature of fluorescent protein coding region to express a fusion protein from pDsRed-EGFPmtag-. Expression of the fusion protein from a single reporter provides a sensitive approach for high-throughput screening of cell-specific PTC events in mixed cell cultures. Results from the read-through analysis of COS7 cells carrying the nonsense mutation pDsRed-EGFPmtag-Y445X treated by PTC 124 showed EGFP transcript level was increased in the COS7 cells treated by PTC124 in a dose-dependent manner. This novel reporter system was applicable to the majority of different PTC patterns and could be used to quantify efficiency of read-through within a single cell or select cells carrying PTC.

Thermally Induced Apoptosis, Necrosis, and Heat Shock Protein Expression in Three-Dimensional Culture

This study was conducted to compare the heat shock responses of cells grown in 2D and 3D culture environments as indicated by the level of heat shock protein 70 expression and the incidence of apoptosis and necrosis of prostate cancer cell lines in response to graded hyperthermia. PC3 cells were stably transduced with a dual reporter system composed of two tandem expression cassettes-a conditional heat shock protein promoter driving the expression of green fluorescent protein (HSPp-GFP) and a cytomegalovirus (CMV) promoter controlling the constitutive expression of a "beacon" red fluorescent protein (CMVp-RFP). Two-dimensional and three-dimensional cultures of PC3 prostate cancer cells were grown in 96-well plates for evaluation of their time-dependent response to supraphysiological temperature. To induce controlled hyperthermia, culture plates were placed on a flat copper surface of a circulating water manifold that maintained the specimens within ±0.1°C of a target temperature. Hyperthermia protocols included various combinations of temperature, ranging from 37°C to 57°C, and exposure times of up to 2 h. The majority of protocols were focused on temperature and time permutations, where the response gradient was greatest. Post-treatment analysis by flow cytometry analysis was used to measure the incidences of apoptosis (annexin V-FITC stain), necrosis (propidium iodide (PI) stain), and HSP70 transcription (GFP expression). Cells grown in 3D compared with 2D culture showed reduced incidence of apoptosis and necrosis and a higher level of HSP70 expression in response to heat shock at the temperatures tested. Cells responded differently to hyperthermia when grown in 2D and 3D cultures. Three-dimensional culture appears to enhance survival plausibly by activating protective processes related to enhanced-HSP70 expression. These differences highlight the importance of selecting physiologically relevant 3D models in assessing cellular responses to hyperthermia in experimental settings.

Live visualization and quantification of pathway signaling with dual fluorescent and bioluminescent reporters

Despite their fundamental importance, the dynamics of signaling pathways in living cells remain challenging to study, due to a lack of non-invasive tools for temporal assessment of signal transduction in desired cell models. Here we report a dual-reporter strategy that enables researchers to monitor signal transduction in mammalian cells in real-time, both temporally and quantitatively. This is achieved by co-expressing green fluorescent protein and firefly luciferase in response to signaling stimuli. To display the versatility of this approach, we constructed and assessed eight unique signaling pathway reporters. We further validated the system by establishing stable NF-κB pathway reporter cell lines. Using these stable cell lines, we monitored the activity of NF-κB-mediated inflammatory pathway in real-time, both visually and quantitatively. Live visualization has the power to reveal individual cell responses and is compatible with single cell analysis, In addition, we provide evidence that this system is readily amenable to a high-throughput format. Together, our findings demonstrate the potential of the dual reporter system, which significantly improves the capacity to study signal transduction pathways in mammalian cells.