Long-term-stable near-infrared polymer dots with ultrasmall size and narrow-band emission for imaging tumor vasculature in vivo.

Fluorescent nanoprobes have become one of the most promising classes of materials for cancer imaging. However, there remain many unresolved issues with respect to the understanding of their long-term colloidal stability and photostability in both biological systems and the environment. In this study, we report long-term-stable near-infrared (NIR) polymer dots for in vivo tumor vasculature imaging. NIR-emitting polymer dots were prepared by encapsulating an NIR dye, silicon 2,3-naphthalocyanine bis(trihexylsilyloxide) (NIR775), into a matrix of polymer dots, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), using a nanoscale precipitation method. The prepared NIR polymer dots were sub-5 nm in diameter, exhibited narrow-band NIR emission at 778 nm with a full width at half-maximum of 20 nm, and displayed a large Stokes shift (>300 nm) between the excitation and emission maxima. In addition, no significant uptake of the prepared NIR polymer dots by either human glioblastoma U87MG cells or human non-small cell lung carcinoma H1299 cells was detected. Moreover, these NIR polymer dots showed long-term colloidal stability and photostability in water at 4 °C for at least 9 months, and were able to image vasculature of xenografted U87MG tumors in living mice after intravenous injection. These results thus open new opportunities for the development of whole-body imaging of mice based on NIR polymer dots as fluorescent nanoprobes.

GRIM-19 opposes reprogramming of glioblastoma cell metabolism via HIF1α destabilization.

The metabolism that sustains cancer cells is adapted preferentially to glycolysis, even under aerobic conditions (Warburg effect). This effect was one of the first alterations in cancer cells recognized as conferring a survival advantage. In this study, we show that gene associated with retinoid-interferon-induced mortality-19 (GRIM-19), which was previously identified as a tumor suppressor protein associated with growth inhibition and cell apoptosis, contributes to the switch between oxidative and glycolytic pathways. In parallel to this, vascular endothelial growth factor, which promotes neovascularization, is also regulated. We have identified hypoxia-inducible factor 1α (HIF1α) as the downstream factor of GRIM-19 in human glioblastoma cell lines. Downregulation of GRIM-19 promotes HIF1α synthesis in a STAT3-dependent manner, which acts as a potential competitive inhibitor for von Hippel-Lindau (pVHL)-HIF1α interaction, and thereby prevents HIF1α from pVHL-mediated ubiquitination and proteasomal degradation. Taken together, it is concluded that GRIM-19, a potential tumor suppressor gene, performs its function in part via regulating glioblastoma metabolic reprogramming through STAT3-HIF1α signaling axis, and this has added new perspective to its role in tumorigenesis, thus providing potential strategies for tumor metabolic therapy.

Function of GRIM-19, a mitochondrial respiratory chain complex I protein, in innate immunity.

Mitochondria respiratory chain (RC), consisting of five multisubunit complexes, is crucial for cellular energy production, reactive oxygen species generation, and regulation of apoptosis. Recently, a few mitochondrial proteins have been reported to be essential for innate immunity, but the function of mitochondrial RC in innate immunity is largely unknown. By knock-out of GRIM-19, a newly identified subunit protein of mitochondrial complex I, in mice, we found that heterogeneous mice (GRIM-19(+/-)) are prone to spontaneous urinary tract infection, mostly by Staphylococcus saprophyticus. Macrophages derived from these mice have compromised mitochondrial complex I activity and increased reactive oxygen species level. Bacterial infection induces a rapid up-regulation of GRIM-19 and complex I activity in the wild-type macrophages, but both are reduced in the macrophages from GRIM-19(+/-) mice. These cells also have decreased intracellular killing ability against S. saprophyticus. The defects for this probably occur in the fusion of bacteria to lysosome, but not in the bacterial engulfment and macrophage migration. In addition, production of proinflammatory cytokines, such as interleukin (IL)-1, IL-12, IL-6, and interferon (IFN)-γ, induced by both bacterial infection and lipopolysaccharide (LPS) and monodansylcadaverine treatment, is also decreased in the GRIM19(+/-) macrophages. Inhibition of mitochondrial RC activity by inhibitors shows a similar reduction on the cytokine production. Due to low cytokine production, the inflammatory response caused by in vivo bacterial challenge in the bladders of GRIM-19(+/-) mice is compromised. This study provides genetic evidence for a critical role of mitochondrial RC in innate immunity.

Depletion of GRIM-19 accelerates hepatocellular carcinoma invasion via inducing EMT and loss of contact inhibition.

UNLABELLED: Genes associated with retinoid-interferon-induced mortality 19 (GRIM-19) was identified as a tumor suppressor protein associated with apoptosis and growth inhibition. Here, we report that the expression levels of GRIM-19 are significantly attenuated in hepatocellular carcinoma (HCC) patients with deteriorating differentiation states, hepatic capsule invasion and microvascular invasion, suggesting the potential role of GRIM-19 not only at the origin but also in the invasive progression of HCCs. To dissect the possible mechanisms by which GRIM-19 regulates tumor cell invasion, we established the hepatic HL-7702 and HCC Huh-7 cell lines stably depleted of GRIM-19. Results show that downregulation of GRIM-19 induces a morphological transformation resembling epithelial-mesenchymal transition (EMT) as well as aberrant expression of epithelial and mesenchymal molecular markers. Additionally, these cells lose contact inhibition, a phenomenon of cessation of cell migration in contact with neighboring cells, as assessed by cell imaging, growth curve and S-phase transition in confluent conditions. CONCLUSION: Our observations demonstrate a novel mechanistic insight into a critical role of GRIM-19 in HCC invasive potential.

Stat3 promotes directional cell migration by regulating Rac1 activity via its activator betaPIX.

Stat3 is a member of the signal transducer and activator of transcription family, which is important for cytokine signaling as well as for a number of cellular processes including cell proliferation, anti-apoptosis and immune responses. In recent years, evidence has emerged suggesting that Stat3 also participates in cell invasion and motility. However, how Stat3 regulates these processes remains poorly understood. Here, we find that loss of Stat3 expression in mouse embryonic fibroblasts leads to an elevation of Rac1 activity, which promotes a random mode of migration by reducing directional persistence and formation of actin stress fibers. Through rescue experiments, we demonstrate that Stat3 can regulate the activation of Rac1 to mediate persistent directional migration and that this function is not dependent on Stat3 transcriptional activity. We find that Stat3 binds to betaPIX, a Rac1 activator, and that this interaction could represent a mechanism by which cytoplasmic Stat3 regulates Rac1 activity to modulate the organization of actin cytoskeleton and directional migration.

Stat3 regulates microtubules by antagonizing the depolymerization activity of stathmin.

Stat3 is a member of the signal transducer and activator of transcription family, which is important in cytokine signaling. Gene ablation studies have revealed a requirement for Stat3 in diverse biological processes (Akira, S. 2000. Oncogene. 19: 2607-2611; Levy, D.E., and C.K. Lee. 2002. J. Clin. Invest. 109:1143-1148). Previously, the function of Stat3 had been attributed exclusively to its transcriptional activity in the nucleus. In this study, we reveal an interaction between Stat3 and the microtubule (MT)-destabilizing protein stathmin. Stathmin did not overtly affect ligand-stimulated Stat3 activation. In contrast, the expression of Stat3 is required for the stabilization of MTs and cell migration. We further demonstrate that Stat3-containing cells are resistant to the MT-destabilizing effect of stathmin overexpression. In addition, down-regulation of stathmin protein levels in Stat3-deficient cells partially reversed the MT and migration deficiencies. Recombinant Stat3 was also capable of reversing stathmin inhibition of tubulin polymerization in vitro. Our results indicate that Stat3 modulates the MT network by binding to the COOH-terminal tubulin-interacting domain of stathmin and antagonizing its MT destabilization activity.

Identification of alternatively spliced GRIM-19 mRNA in kidney cancer tissues.

Gene associated with Retinoid-Interferon-induced Mortality (GRIM)-19 was originally identified as a regulatory gene necessary for interferon-beta and retinoic acid-induced cell death. Further studies revealed that GRIM-19 is a subunit of mitochondrial respiratory chain complex I. Previous studies show that the expression of GRIM-19 is lost or severely depressed in a number of primary renal cell carcinomas (RCCs) and in some urinogenital tumors. Four point mutations were found in the GRIM-19 gene in mitochondria-rich (Hürthle) tumors and one point mutation was reported in RCC. In this study, we report an alternatively splicing form of GRIM-19 mRNA with intron 3 by reverse transcriptase PCR. This splicing variant is found in kidney tumor tissues but not in matched normal tissues. Furthermore, we found that in addition to GRIM-19, the protein level of NDUFS3, which is another mitochondrial complex I subunit, was also diminished in kidney tumor tissues when compared with paired normal tissues. Our finding suggested that the alternative splicing form of GRIM-19 is tumor tissue specific.

The mitochondrial respiratory chain controls intracellular calcium signaling and NFAT activity essential for heart formation in Xenopus laevis.

The mitochondrial respiratory chain (MRC) plays crucial roles in cellular energy production. However, its function in early embryonic development remains largely unknown. To address this issue, GRIM-19, a newly identified MRC complex I subunit, was knocked down in Xenopus laevis embryos. A severe deficiency in heart formation was observed, and the deficiency could be rescued by reintroducing human GRIM-19 mRNA. The mechanism involved was further investigated. We found that the activity of NFAT, a transcription factor family that contributes to early organ development, was downregulated in GRIM-19 knockdown embryos. Furthermore, the expression of a constitutively active form of mouse NFATc4 in these embryos rescued the heart developmental defects. NFAT activity is controlled by a calcium-dependent protein phosphatase, calcineurin, which suggests that calcium signaling may be disrupted by GRIM-19 knockdown. Indeed, both the calcium response and calcium-induced NFAT activity were impaired in the GRIM-19 or NDUFS3 (another complex I subunit) knockdown cell lines. We also showed that NFAT can rescue expression of Nkx2.5, which is one of the key genes for early heart development. Our data demonstrated the essential role of MRC in heart formation and revealed the signal transduction and gene expression cascade involved in this process.

NFAT directly regulates Nkx2-5 transcription during cardiac cell differentiation.

BACKGROUND INFORMATION: The transcription factor NFAT (nuclear factor of activated T-cell) family comprises important regulators in immuno-responses and mouse embryonic development, including early cardiovascular and heart valve development. The mechanism involved, however, is not fully understood. Nkx2-5 (NK2 transcription factor related, locus 5) is one of the earliest genes expressed in early cardiac progenitor cells and is essential for heart tube development by control of a subset of cardiac muscle-specific genes. Previously we found that downregulation of mitochondrial respiratory chain complex I caused severe cardiac deficiencies during heart tube development in Xenopus embryos associated with compromised Nkx2-5 expression. However, the heart defects and Nkx2-5 expression could be rescued by a constitutively activated NFAT, suggesting a possible link between NFAT and Nkx2-5 during early heart development. RESULTS: In the present study, we demonstrate that NFAT regulates Nkx2-5 expression in both mouse ES (embryonic stem) cells and P19 cells, a mouse model for embryonic differentiation. We found that there are six core NFAT-binding elements in the 5' regulatory region of the Nkx2-5 gene. Although NFAT is able to bind directly to all but one of these elements, it activates Nkx2-5 transcription only via a specific binding site in the distal enhancer region. Interestingly, the transcriptional activity of NFAT is largely dependent on the co-factor GATA (GATA-binding transcription factor), which binds to an element adjacent to this key NFAT-binding site. Furthermore, binding of the endogenous NFAT to this particular site was observed during cardiac differentiation in mouse ES and P19 cells. CONCLUSIONS: The results suggest that Nkx2-5 is a direct target of NFAT that co-ordinates with other transcription factors such as GATA4 to regulate Nkx2-5 during cardiogenesis.

SCG10-like protein (SCLIP) is a STAT3-interacting protein involved in maintaining epithelial morphology in MCF-7 breast cancer cells.

STAT (signal transducer and activator of transcription) 3 is a key contributor to cancer cell migration and invasion, with excessive STAT3 activity promoting growth arrest, cell-cell dissociation and increased migration of breast cancer epithelial cells. The STAT3-regulated mechanisms involved in this process, however, are not fully defined. Previously, we had revealed SCLIP [SCG10 (superior cervical ganglia protein 10)-like protein] as a novel STAT3-interacting protein. In the present study, we show that STAT3 binds the C-terminal tubulin-associating region of SCLIP. In a search for a function of SCLIP, we show that SCLIP was down-regulated during OSM (oncostatin M) treatment in MCF-7 cells, which also stimulates epithelial morphology loss. SCLIP knockdown likewise triggered a loss of epithelial morphology which included reduced E-cadherin expression. We found that STAT3 was required to maintain SCLIP stability. Furthermore, inhibition of OSM-induced STAT3 activity preserved SCLIP expression and MCF-7 epithelial monolayers. Taken together, we propose that a STAT3-SCLIP interaction is required to preserve SCLIP stability and contributes to the maintenance of normal epithelial morphology. Disruption of the STAT3-SCLIP interaction with OSM may contribute to cytokine-mediated loss in cell-cell attachment and morphology transition in MCF-7 cells.

Oct4 is expressed in human gliomas and promotes colony formation in glioma cells.

There is increasing evidence that self-renewal capacity of cancer cells is critical for carcinogenesis; hence, it is vital to examine the expression and involvement of self-renewal regulatory genes in these cells. Here, we reported that Oct4, a well-known regulator of self-renewal in embryonic stem cells, was highly expressed in human gliomas and glioma cell lines, and the expression levels were increased in parallel with increasing glioma grades. In in vitro cell cultures, Oct4 was only expressed in rat C6 glioma cells and rat neural stem cells but not in rat brain differentiated cells. Downregulation of Oct4 expression by RNA interference in C6 cells was associated with reduced cell proliferation and colony formation. Further analysis revealed that Oct4 could upregulate phosphorylation of Stat3 to promote tumor cell proliferation. Overexpression of Oct4 in C6 cells increased the expression of nestin but decreased the expression of GFAP suggesting that Oct4 might inhibit the differentiation of glioma cells. Our findings may provide further evidence for the stem cell theory of carcinogenesis. In contrast, the results might also imply that Oct4 contributes to the existence of undifferentiated cells in gliomas.

NF-kappaB p52, RelB and c-Rel are transported into the nucleus via a subset of importin alpha molecules.

In resting cells NF-kappaB transcription factors are retained in the cytoplasm as latent inactive complexes, until they are activated and rapidly transported into the nucleus. We show that all NF-kappaB proteins are imported into the nucleus via a subset of importin alpha isoforms. Our data indicate that the NF-kappaB components of the classical and alternative pathways have somewhat different specifities to importin alpha molecules. Based on the results from binding experiments of in vitro-translated and Sendai virus infection-induced or TNF-alpha-stimulated endogenous NF-kappaB proteins, it can be predicted that the specifity of NF-kappaB proteins to importin alpha molecules is different and changes upon the composition of the imported dimer. p52 protein binds directly to importin alpha3, alpha4, alpha5 and alpha6 and c-Rel binds to importin alpha5, alpha6 and alpha7 via a previously described monopartite nuclear localization signals (NLSs). Here we show that RelB, instead, has a bipartite arginine/lysine-rich NLS that mediates the binding of RelB to importin alpha5 and alpha6 and subsequent nuclear translocation of the protein. Moreover, we show that the nuclear import of p52/RelB heterodimers is mediated exclusively by the NLS of RelB. In addition, we found that the NLS of p52 mediates the nuclear import of p52/p65 heterodimers.

Preparation and properties of OSA-modified taro starches and their application for stabilizing Pickering emulsions.

In this study, the octenylsuccinylated taro starches (OSTS) with different degree of substitution (DS, from 0.009 to 0.032) were prepared and their structural properties such as granule size, wettability and morphology were studied. The purpose of this work was to elucidate the OSTS with different DS using as particle stabilizers for Pickering emulsions, and the effect of DS on the stability, droplet size, microstructure and rheological properties of OSTS-stabilized emulsions were investigated. Octenylsuccinic anhydride (OSA) modification had slight effects on the morphology or granule size of taro starch, but markedly increased the contact angle from 25.4° to 70.1°. Octenylsuccinylation significantly improved the emulsifying capacity of taro starch granules, and thus OSTS-stabilized emulsions formed at higher DS exhibited better stability. Droplet size distribution results and microscopic observations revealed that OSTS-emulsion prepared at DS of 0.032 had the smallest droplet size and most uniform distribution compared with the other emulsions. The rheological results indicated that both OSTS-emulsions (DS, from 0.009 to 0.032) showed shear-thinning behavior as a non-Newtonian fluid, and the viscosities of emulsions were progressively improved with the increase of DS. Moreover, the G' and G″ values of OSTS-emulsions increased with increasing DS, reflecting the enhanced viscoelastic properties and exhibiting an improved rigidity of the emulsions. The above results suggested that higher-DS favored the formation of superior OSTS-emulsions, and thus OSTS with a high DS (DS ≥ 0.018) can be used for preparing stable Pickering emulsions.

The LIM/homeodomain protein Islet1 recruits Janus tyrosine kinases and signal transducer and activator of transcription 3 and stimulates their activities.

Islet1 (Isl1) belongs to the LIM homeodomain transcription factor family. Its roles in differentiation of motor neurons and organogenesis of pancreas and heart have been revealed. However, less is known about its regulatory mechanism and the target genes. In this study, we identified interactions between Isl1 and Janus tyrosine kinase (JAK), as well as signal transducer and activator of transcription (Stat)3, but not Stat1 and Stat5, in mammalian cells. We found that Isl1 not only forms a complex with Jak1 and Stat3 but also triggers the tyrosine phosphorylation of Jak1 and its kinase activity, thereby elevating the tyrosine phosphorylation, DNA binding activity, and target gene expression of Stat3. In vivo, the tyrosine-phosphorylated Stat3 was colocalized with Isl1 in the nucleus of the mouse motor neurons in spinal cord after nerve injury. Correspondingly, electroporation of Isl1 and Stat3 into the neural tube of chick embryos resulted in the activation of a reporter gene expression controlled by a Stat3 regulatory sequence, and cotransfection of Isl1 and Stat3 promoted the proliferation of the mouse motor neuron cells. Our data suggest a novel role of Isl1 as an adaptor for Jak1 and Stat3 and reveal a possible functional link between LIM homeodomain transcription factors and the Jak-Stat pathway.

Regulation of Stat3 nuclear import by importin alpha5 and importin alpha7 via two different functional sequence elements.

Regulated import of STAT proteins into the nucleus through the nuclear pores is a vital event. We previously identified Arg214/215 in the coiled-coil domain and Arg414/417 in the DNA binding domain involved in the ligand-induced nuclear translocation of Stat3. In this study, we investigated the mechanism for Stat3 nuclear transport. We report here that among five ubiquitously expressed human importin alphas, importin alpha5 and alpha7, but not importin alpha1, alpha3, and alpha4, bind to Stat3 upon cytokine stimulation. Similar results were observed for Stat1, but not for Stat5a and 5b, which were unable to interact with any of the importin alphas. The C-terminus of importin alpha5 is necessary but not sufficient for Stat3 binding. Truncation mutant of Stat3 (aa1-320) that contains Arg214/215 exhibits specific binding to importin alpha5, and an exclusive nuclear localization. Point mutations of Arg214/215 in this mutant destroy importin alpha5 binding and its nuclear localization. In contrast, the truncation mutant (aa320-770) including Arg414/417 fails to interact with importin alpha5 and is localized in the cytoplasm. However, both sequence elements are necessary for the full-length Stat3's interaction with importin alpha5. These results suggest that Arg214/215 is likely the binding site for importin alpha5, whereas Arg414/417 may not be involved in the direct binding, but necessary for maintaining the proper conformation of Stat3 dimer for importin binding. A model for Stat3 nuclear translocation is proposed based on these data.

GRIM-19, a cell death regulatory protein, is essential for assembly and function of mitochondrial complex I.

Mitochondria play essential roles in cellular energy production via the oxidative phosphorylation system (OXPHOS) consisting of five multiprotein complexes and also in the initiation of apoptosis. NADH:ubiquinone oxidoreductase (complex I) is the largest complex that catalyzes the first step of electron transfer in the OXPHOS system. GRIM-19 was originally identified as a nuclear protein with apoptotic nature in interferon (IFN)- and all-trans-retinoic acid (RA)-induced tumor cells. To reveal its biological role, we generated mice deficient in GRIM-19 by gene targeting. Homologous deletion of GRIM-19 causes embryonic lethality at embryonic day 9.5. GRIM-19(-/-) blastocysts show retarded growth in vitro and, strikingly, display abnormal mitochondrial structure, morphology, and cellular distribution. We reexamined the cellular localization of GRIM-19 in various cell types and found its primary localization in the mitochondria. Furthermore, GRIM-19 is detected in the native form of mitochondrial complex I. Finally, we show that elimination of GRIM-19 destroys the assembly and electron transfer activity of complex I and also influences the other complexes in the mitochondrial respiratory chain. Our result demonstrates that GRIM-19, a gene product with a specific role in IFN-RA-induced cell death, is a functional component of mitochondrial complex I and is essential for early embryonic development.

Scalable Production of Mechanically Robust Antireflection Film for Omnidirectional Enhanced Flexible Thin Film Solar Cells.

Antireflection (AR) at the interface between the air and incident window material is paramount to boost the performance of photovoltaic devices. 3D nanostructures have attracted tremendous interest to reduce reflection, while the structure is vulnerable to the harsh outdoor environment. Thus the AR film with improved mechanical property is desirable in an industrial application. Herein, a scalable production of flexible AR films is proposed with microsized structures by roll-to-roll imprinting process, which possesses hydrophobic property and much improved robustness. The AR films can be potentially used for a wide range of photovoltaic devices whether based on rigid or flexible substrates. As a demonstration, the AR films are integrated with commercial Si-based triple-junction thin film solar cells. The AR film works as an effective tool to control the light travel path and utilize the light inward more efficiently by exciting hybrid optical modes, which results in a broadband and omnidirectional enhanced performance.

Production of vinegar from purple sweet potato in a liquid fermentation process and investigation of its antioxidant activity.

Acetobacter pasteurianus JST-S was screened from solid fermented grains of vinegar in China, identified by molecular analysis, and used for the production of purple sweet potato vinegar using purple sweet potato as the substrate. By orthogonal experiment, maximum total acid concentration (4.26% [v/v]) was achieved under optimized conditions as follows: fermentation time, 3.5 days; ethanol content, 9% v/v; and inoculum size, 8% v/v. During the production of purple potato vinegar, the anthocyanin concentration decreased from 652.07 to 301.73 µg/mL. The antioxidant activity of products, including diphenyl-picryl hydrazide radical-scavenging capacity (above 60%), reducing power (above 0.47), and hydroxyl radical-scavenging capacity (above 46%), showed positive linear regression (P < 0.01), which could be related with the changes in anthocyanin concentration and antioxidant activities at different stages of vinegar fermentation. The acetic acid and other non-phenolic antioxidants in purple sweet potato vinegar may have contributed to the antioxidant activities. Results of these studies may provide a reference for the industrial production of vinegar by liquid fermentation of purple sweet potato.

Increased dosage of Runx1/AML1 acts as a positive modulator of myeloid leukemogenesis in BXH2 mice.

The RUNX1/AML1 gene on chromosome 21 is most frequently inactivated in human leukemias. In addition, an increased dose of RUNX1 is suggested as a basis for several kinds of leukemias. Amplifications of chromosome 21 or the RUNX1 gene are shown to be associated with leukemias with lymphoid lineage, whereas its involvement in myeloid lineage remains unclear. In this study, we generated GATA-1 promoter-driven Runx1 transgenic (Tg) mice, which showed a transient mild increase of megakaryocyte marker-positive myeloid cells but no spontaneous leukemia. These mice were then crossed with BXH2 mice, which have a replication-competent retrovirus in the mouse and develop myeloid leukemia due to insertional mutagenesis by random integration of the virus. Overexpressed Runx1 transgene in BXH2 mice resulted in shortening of the latency of leukemia with increased frequency of megakaryoblastic leukemia, suggesting that increased Runx1 dosage is leukemogenic in myeloid lineage. Identifications of retroviral integration sites revealed the genetic alterations that may cooperate with Runx1 overdose in myeloid leukemogenesis. This mouse model may be useful for analysing the pathogenesis of myeloid leukemias with RUNX1 overdose, especially to examine whether an extra-copy of RUNX1 by trisomy 21 is causally related to Down's syndrome-related acute megakaryoblastic leukemia (DS-AMKL).

Structure, function, and regulation of STAT proteins.

The Signal Transducer and Activator of Transcription (STAT) family of proteins was first discovered in the 1990's as key proteins in cytokine signaling. Since then, the field has greatly advanced in the past 15 years, providing significant insight into the structure, function, and regulation of STATs. STATs are latent cytoplasmic transcription factors consisting of seven mammalian members. They are Tyr phosphorylated upon activation, a post-translational modification critical for dimerization, nuclear import, DNA binding, and transcriptional activation. In recent years, unphosphorylated STATs have also been observed to dimerize and drive transcription, albeit by yet an obscure mechanism. In addition, the function of cytoplasmic STATs is beginning to emerge. Here, we describe the structure, function, and regulation of both unphosphorylated and phosphorylated STATs. STAT isoforms from alternative splicing or proteolytic processing, and post-translational modifications affecting STAT activities are also discussed.