NIH 3T3 Mouse Fibroblast Cells Research Articles

Identification of a novel Wnt5a-CK1ε-Dvl2-Plk1-mediated primary cilia disassembly pathway

Non-motile primary cilium is an antenna-like structure whose defect is associated with a wide range of pathologies, including developmental disorders and cancer. Although mechanisms regulating cilia assembly have been extensively studied, how cilia disassembly is regulated remains poorly understood. Here, we report unexpected roles of Dishevelled 2 (Dvl2) and interphase polo-like kinase 1 (Plk1) in primary cilia disassembly. We demonstrated that Dvl2 is phosphorylated at S143 and T224 in a manner that requires both non-canonical Wnt5a ligand and casein kinase 1 epsilon (CK1ɛ), and that this event is critical to interact with Plk1 in early stages of the cell cycle. The resulting Dvl2-Plk1 complex mediated Wnt5a-CK1ɛ-Dvl2-dependent primary cilia disassembly by stabilizing the HEF1 scaffold and activating its associated Aurora-A (AurA), a kinase crucially required for primary cilia disassembly. Thus, via the formation of the Dvl2-Plk1 complex, Plk1 plays an unanticipated role in primary cilia disassembly by linking Wnt5a-induced biochemical steps to HEF1/AurA-dependent cilia disassembly. This study may provide new insights into the mechanism underlying ciliary disassembly processes and various cilia-related disorders.

Immortalization and malignant transformation of Eukaryotic cells

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.

Immobilization of gelatin onto natural nanofibers for tissue engineering scaffold applications without utilization of any crosslinking agent

Bacterial cellulose was oxidized by periodate oxidation to give rise to 2,3-dialdehyde bacterial cellulose (DABC) with 60.3 ± 0.5 % aldehyde content, which was further reacted with gelatin (Gel) for the immobilization of Gel to form DABC/Gel nanocomposites. The scanning electron microscopy and transmission electron microscopy revealed that DABC/Gel exhibited the refined 3D nano-network structures and the average thickness of Gel coatings in the composites was about 75 nm. FTIR and XPS were utilized to analyze the functional groups and chemical states of DABC/Gel nanocomposites. The results inferred that Gel was fixed on DABC nanofibers via the Schiff base reaction between –NH2 in Gel and –CHO in DABC backbone. NIH3T3 mice fibroblast cells were used for determining the cytocompatibility of the scaffolds. The morphology of the cells was observed through optical inverted microscopy. The results show that DABC/Gel can be used as scaffold material in tissue engineering.

Nuclear Smad7 Overexpressed in Mesenchymal Cells Acts as a Transcriptional Corepressor by Interacting with HDAC-1 and E2F to Regulate Cell Cycle

SummarySmad family proteins are essential intracellular mediators that regulate transforming growth factor-β (TGF-β) ligand signaling. In response to diverse stimuli, Smad7 is rapidly expressed and acts as a cytoplasmic inhibitor that selectively interferes with signals elicited from TGF-β family receptors. In addition, earlier works have indicated that retrovirally transduced Smad7 induces long-lasting cell proliferation arrest in a variety of mesenchymal cells through down-regulation of G1 cyclins. However, the molecular mechanisms underlying the cytostatic effects of Smad7 remain unknown. We show here that Smad7 can form a complex with endogenous histone deacetylase proteins HDAC-1 and HDAC-3 in NIH 3T3 mouse fibroblast cells. By contrast, forced expression of a dominant-negative variant of HDAC-1 efficiently protected cells against Smad7 proliferation inhibition, suggesting that Smad7 depends on the deacetylase activity of its associated HDAC-1 to arrest the cell cycle. Furthermore, Smad7 caused HDAC-1 bind to E2F-1 to form a ternary complex on chromosomal DNA containing an E2F-binding motif and leading to repression in the activity of the E2F target genes. Smad7 mutations that prevented its binding to either HDAC-1 or E2F-1 resulted in a significant decrease in Smad7-mediated inhibition of cell proliferation. The present results strongly suggest that nuclear Smad7 is a transcriptional corepressor for E2F, providing a molecular basis for the Smad7-induced arrest of the cell cycle.

Stimuli-responsive hydrogels for controlled pilocarpine ocular delivery

A series of vinyl hydrogels containing α-aminoacid (l-histidine, l-valine) residues was synthesized and their swelling properties evaluated at different pHs and temperatures. Unlike the zwitterionic compound containing only the l-histidine, a dual-stimuli responsiveness was improved in the carboxyl acid hydrogels carrying the l-valine residues (HVa). Besides the COOH functionality, the presence of either isopropyl and amido groups in the monomer structure renders the hydrogel also temperature-responsive, in a similar manner as the well-known poly(N-isopropylacrylamide) (pNIPAAm). The three HVa hydrogels (cross-linked with 1, 2, and 5mol% of N,N′-ethylene-bisacrylamide, EBA) show a phase separation at the same critical pH4, although a different swelling was improved by the amount of EBA. In buffered solutions, the effect of increasing temperature led to decrease the swelling and, as the pH is close to the critical one, a further and sharper collapse of the hydrogel may be tuned. The release study of pilocarpine in physiological conditions showed a burst effect within the first few hours, followed by a sustained release for a week. The initial burst effect was strongly dependent on the kind of hydrogel investigated. As the pilocarpine is a basic molecule (pKa7.2), it may interact more strongly with the free carboxyl groups in the ionized state of the HVa hydrogels than the zwitterionic species of the histidine compounds. The releasing profile shows a three time greater release of the pilocarpine loaded in the HVa hydrogels. The hydrogels were found to be non-toxic against the mouse fibroblast NIH3T3 cells. The presence of pilocarpine strongly increased the cell proliferation even after 2days.

Manipulation of NIH3T3 cells with functionalized single-walled carbon nanotubes under a magnetic field

We demonstrated the magnetic manipulation of NIH3T3 mouse fibroblast cells transfected with purified single-walled carbon nanotubes (SWNTs). The SWNTs were functionalized by using simple acid treatment. Introducing defects and adding functional groups into the SWNTs increased the solubility and induced ferromagnetic behavior in the functionalized SWNTs (fSWNTs). The uptake of the fSWNTs was confirmed by transmission electron microscopy (TEM). Cytotoxicity was reduced because of the removal of metallic impurities during functionalization. Cells with soluble magnetic fSWNTs were attracted and aligned in the direction of the magnetic source. Our findings may lead to improvement in non-invasive and non-toxic drug delivery as well as targeted cell therapy with fSWNTs.

A Small Molecule Mimicking a Phosphatidylinositol (4,5)-Bisphosphate Binding Pleckstrin Homology Domain

Inositol phospholipids have emerged as important key players in a wide variety of cellular functions. Among the seven existing inositol phospholipids, phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) has attracted much attention in recent years due to its important role in numerous cellular signaling events and regulations, which in turn impact several human diseases. This particular lipid is recognized in the cell by specific lipid binding domains, such as the Pleckstrin-homology (PH) domain, which is also employed as a tool to monitor this important lipid. Here, we describe the synthesis and biological characterization of a small molecule that mimics the PH domain as judged by its ability to bind specifically to only PI(4,5)P(2) and effectively compete with the PH domain in vitro and in a cellular environment. The binding constant of this small molecule PH domain mimetic (PHDM) was determined to be 17.6 ± 10.1 μM, similar in potency to the PH domain. Using NIH 3T3 mouse fibroblast cells we demonstrated that this compound is cell-permeable and able to modulate PI(4,5)P(2)-dependent effects in a cellular environment such as the endocytosis of the transferrin receptor, loss of mitochondria, as well as stress fiber formation. This highly PI(4,5)P(2)-specific chemical mimetic of a PH domain not only is a powerful research tool but might also be a lead compound in future drug developments targeting PI(4,5)P(2)-dependent diseases such as Lowe syndrome.

A pharmacological cocktail for arresting actin dynamics in living cells

The actin cytoskeleton is regulated by factors that influence polymer assembly, disassembly, and network rearrangement. Drugs that inhibit these events have been used to test the role of actin dynamics in a wide range of cellular processes. Previous methods of arresting actin rearrangements take minutes to act and work well in some contexts, but can lead to significant actin reorganization in cells with rapid actin dynamics, such as neutrophils. In this paper, we report a pharmacological cocktail that not only arrests actin dynamics but also preserves the structure of the existing actin network in neutrophil-like HL-60 cells, human fibrosarcoma HT1080 cells, and mouse NIH 3T3 fibroblast cells. Our cocktail induces an arrest of actin dynamics that initiates within seconds and persists for longer than 10 min, during which time cells maintain their responsivity to external stimuli. With this cocktail, we demonstrate that actin dynamics, and not simply morphological polarity or actin accumulation at the leading edge, are required for the spatial persistence of Rac activation in HL-60 cells. Our drug combination preserves the structure of the existing cytoskeleton while blocking actin assembly, disassembly, and rearrangement, and should prove useful for investigating the role of actin dynamics in a wide range of cellular signaling contexts.

Caffeine lengthens circadian rhythms in mice

Although caffeine alters sleep in many animals, whether or not it affects mammalian circadian clocks remains unknown. Here, we found that incubating cultured mammalian cell lines, human osteosarcoma U2OS cells and mouse fibroblast NIH3T3 cells, with caffeine lengthened the period of circadian rhythms. Adding caffeine to ex vivo cultures also lengthened the circadian period in mouse liver explants from Per2::Luciferase reporter gene knockin mice, and caused a phase delay in brain slices containing the suprachiasmatic nucleus (SCN), where the central circadian clock in mammals is located. Furthermore, chronic caffeine consumption ad libitum for a week delayed the phase of the mouse liver clock in vivo under 12 h light–dark conditions and lengthened the period of circadian locomotor rhythms in mice under constant darkness. Our results showed that caffeine alters circadian clocks in mammalian cells in vitro and in the mouse ex vivo and in vivo.

Regulation of Matrix Metalloproteinases and Invasion by G<SUB>α12/13</SUB> Proteins in NIH3T3 Mouse Fibroblast Cells

The G12 subfamily of the heterotrimeric G proteins, Galpha12 and Galpha13, has been implicated as an important signaling component in various cellular processes including oncogenesis and cells invasion. Our previous report showed that the expression of an activated mutant of Galpha12 (Galpha12QL) or Galpha13 (Galpha13QL) leads to cell invasion in MCF10A human breast epithelial cells. The present study aimed to investigate the role of Galpha12 and Galpha13 in the malignant phenotypic conversion of NIH3T3 mouse fibroblast cells. Galpha12QL and Galpha13QL induced an invasive phenotype in NIH3T3 cells. In addition, the activation of Galpha12 and Galpha13 upregulated matrix metalloproteinase (MMP)-2 while MMP-9 was not affected by either Galpha12QL or Galpha13QL. Using female NOD/SCID mice injected with NIH3T3 cells stably expressing Galpha12QL, we provided in vivo confirmation of Galpha12-mediated MMP-2 upregulation. Taken together, this study elucidated the role of Galpha12/13 in regulating malignant phenotypic conversion of NIH3T3 fibroblast cells, validating the role of Galphal2/13 in tumorigenesis.

In vitro cytotoxicity and phototoxicity study of cosmetics colorants

The aim of the work was early identification of preventable risk factors connected with the consumers usage of products of everyday use, such as cosmetics, toys and children products, and other materials intended for contact with human skin. The risk factor is represented by substances with irritation potential and subsequent possible sensitisation, resulting in negative impact on human physical and psychical health with social and societal consequences. The legislation for cosmetics, chemical substances and other products requires for hazard identification the application of alternative toxicological methods in vitro without the use of animals. For this reason we used a battery of alternative assays in vitro, based on cell cultures. Progressive methods of molecular biology, based on fluorimetry and fluorescence, were employed for identification of early morphological and functional changes on cellular level. Four colorants frequently used in cosmetics (P-WS Caramel, Chlorophyllin, Unicert Red K 7054-J and Unicert Red K 7008-J) were tested on cell line NIH3T3 (mouse fibroblast cell) and 3T3 Balb/c with/without UV irradiation (dose 5 J cm −2). Fluorescence methods for the study of cell damage using fluorescence probes offer results for the evaluation of cytotoxicity and cell viability of adherent cells. We detected intracellular production of ROS investigated by molecular probe CM-H 2DCFDA, which is primarily sensitive to the increased production of hydrogen peroxide or its downstream products. Toxic effects on the cellular level were identified by viability tests using Neutral Red uptake and MTT assay, where the live cells reduce yellow soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan crystals. The reaction was investigated on mitochondrial membrane of living cells and the type of cell death was determined using Apoptosis detection kit. Cytotoxicity tests revealed health risks of using Chlorophyllin and Unicert Red K 7054-J.

Polymeric Multilayers that contain Silver Nanoparticles can be Stamped onto Biological Tissues to Provide Antibacterial Activity.

We report the design of polyelectrolyte multilayers (PEMs) that can be prefabricated on an elastomeric stamp and mechanically transferred onto biomedically-relevant soft materials, including medical-grade silicone elastomers (E'~450-1500 kPa; E'-elastic modulus) and the dermis of cadaver-skin (E'~200-600 kPa). Whereas initial attempts to stamp PEMs formed from poly(allylamine hydrochloride) and poly(acrylic acid) resulted in minimal transfer onto soft materials, we report that integration of micrometer-sized beads into the PEMs (thicknesses of 6-160 nm) led to their quantitative transfer within 30 seconds of contact at a pressure of ~196 kPa. To demonstrate the utility of this approach, PEMs were impregnated with a range of loadings of silver-nanoparticles and stamped onto the dermis of human cadaver-skin (a wound-simulant) that was subsequently incubated with bacterial cultures. Skin-dermis stamped with PEMs that released 0.25±0.01 μg cm-2 of silver ions caused a 6 log10 reduction in colony forming units of Staphylococcus epidermidis and Pseudomonas aeruginosa within 12 h. Significantly, this level of silver release is below that which is cytotoxic to NIH 3T3 mouse fibroblast cells. Overall, this study describes a general and facile approach for the functionalization of biomaterial surfaces without subjecting them to potentially deleterious processing conditions.

H-ras localizes to cell nuclei and varies with the cell cycle.

H-Ras functions as a signal switch molecule in numerous signaling pathways in the cytoplasm, requiring H-Ras localization to the inner surface of the cytoplasmic membrane, and H-Ras is considered to be a cytoplasmic protein. Immunoblot studies of cells transformed by overexpression of c-H-ras indicated that H-Ras protein was present in both cytoplasmic and nuclear extracts, suggesting a possible correlation of nuclear H-Ras and cellular transformation. Unexpectedly, additional studies revealed that H-Ras protein was also present in the nuclei of nontransformed and primary mouse cells, which do not overexpress H-Ras. Mouse fibroblast NIH 3T3 cells, L cells, and a primary fibroblast line all had H-Ras present in both cytoplasmic and nuclear extracts. Nuclear extracts of cells synchronized by growth without serum displayed an increasing amount of H-Ras and cyclin D1 as cells grew after serum addition. Treatment with farnesyltransferase inhibitor caused loss of H-Ras from the nucleus. Immunofluorescence in situ studies of nuclei from synchronized cultures showed that H-Ras protein appeared in and disappeared from the nuclei as the cells moved through the growth cycle. This cycling occurred in both nontransformed and ras-transformed cells. Flow cytometry measurements on parallel cultures revealed that the time point at which the greatest percentage of cells were in S phase, for each line, corresponded to appearance of a noticeably stronger in situ signal for H-Ras. H-Ras may participate in nuclear signaling pathways associated with replication in addition to its cytoplasmic signaling functions.

Inhibiting scar formation in vitro and in vivo by adenovirus‐mediated mutant Smad4: a preliminary report

The best characterized signalling pathway employed by transforming growth factor-beta (TGF-β) is the Smad pathway. We focused on Smad4, because it is essential for the activation of Smad-dependent target genes. We aimed to explore the possibility of inhibiting scar formation after wounding by blocking TGF-β signalling by means of a gene therapy approach using adenovirus-mediated expression of mutant Smad4. The coding sequence of the dominant-negative mutant Smad4ΔM4, which has a deletion in the linker region of Δ275-322, was introduced by homologous recombination into an adenovirus vector to generate the recombinant vector Ad-ΔM4, which encoded Smad4ΔM4. Mouse fibroblast NIH 3T3 cells were transfected with Ad-ΔM4 and cell proliferation, collagen protein production, and the expression of collagen type I and type III mRNA were evaluated in vitro using a cell proliferation test, western blot analysis, and RT-PCR, respectively. Cell proliferation and the expression of collagen type I and type III mRNA and protein were all inhibited by the transfection of Ad-ΔM4. In vivo, Ad-ΔM4 was applied externally to wounds on rats, and histological examination and quantification of the scars were performed to evaluate the curative effect. The transfection of Ad-ΔM4 successfully inhibited scar formation in rat wounds. In conclusion, Ad-ΔM4 can block the TGF-β signalling of mouse wound cells effectively. In addition, gene therapy with Ad-ΔM4 can effectively inhibit wound scarring in rats and may potentially be applied to clinical treatment of scars.

Plasmid DNA-loaded chitosan/TPP nanoparticles for topical gene delivery

Topical application of plasmid DNA represents an attractive route of gene delivery. Although chitosan (CS) has been widely investigated as a gene-carrier, there is very limited information about the skin application of CS-based systems for DNA. This study evaluated pDNA-loaded chitosan nanoparticles (CS-NPs) for skin gene delivery. NPs were prepared by inducing the gelation of CS upon interaction with sodium tripolyphosphate. pSV-β-Gal was used as a reporter gene. The size, surface charge, and the other in vitro characteristics of CS-NPs were examined. Primary human dermal fibroblast cells (HDF) and mouse fibroblast NIH 3T3 cell lines (ATCC CCL-92) were used for in vitro transfection studies. In in vivo study, CS-NPs were applied to the skin of baby and adult Sprague Dawley rats by spreading on the shaved area of the back of animals. During a week animals were sacrificed and skin biopsies were taken for β-Gal expression. β-galactosidase enzyme activity was determined spectrophotometrically at 420 nm. The distribution of β-galactosidase expressing cells within the skin tissue was observed by X-gal histochemical method. β-galactosidase was continuously expressed at the nanoparticle-treated skin during the 7 days. High and continuous β-Gal expressions were obtained with CS-NPs, although it was low in the first day. When a comparison was made between the data of baby and adult rats, markedly high transfection were measured in the skin samples of the baby rats. NPs protected pDNA against the enzyme and serum attacks. In conclusion, CS-NPs showed in vivo transfection potential in rats for skin gene delivery.

Synthesis and Photophysical Properties of Thioglycosylated Chlorins, Isobacteriochlorins, and Bacteriochlorins for Bioimaging and Diagnostics

The facile synthesis and photophysical properties of three nonhydrolyzable thioglycosylated porphyrinoids are reported. Starting from meso-perfluorophenylporphyrin, the nonhydrolyzable thioglycosylated porphyrin (PGlc₄), chlorin (CGlc₄), isobacteriochlorin (IGlc₄), and bacteriochlorin (BGlc₄) can be made in 2-3 steps. The ability to append a wide range of targeting agents onto the perfluorophenyl moieties, the chemical stability, and the ability to fine-tune the photophysical properties of the chromophores make this a suitable platform for development of biochemical tags, diagnostics, or as photodynamic therapeutic agents. Compared to the porphyrin in phosphate buffered saline, CGlc₄ has a markedly greater absorbance of red light near 650 nm and a 6-fold increase in fluorescence quantum yield, whereas IGlc₄ has broad Q-bands and a 12-fold increase in fluorescence quantum yield. BGlc₄ has a similar fluorescence quantum yield to PGlc₄ (<10%), but the lowest-energy absorption/emission peaks of BGlc₄ are considerably red-shifted to near 730 nm with a nearly 50-fold greater absorbance, which may allow this conjugate to be an effective PDT agent. The uptake of CGlc₄, IGlc₄, and BGlc₄ derivatives into cells such as human breast cancer cells MDA-MB-231 and K:Molv NIH 3T3 mouse fibroblast cells can be observed at nanomolar concentrations. Photobleaching under these conditions is minimal.

Alteration of nuclear protein profiling for NIH‐3T3 cells exposed to H2O2

Oxidative stress is a threat to mammalian cells. To better understand the molecular response and mechanism underlying oxidative stress, we applied two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-fight mass spectrometry analysis to identify differential nuclear protein profiling of mouse fibroblast NIH-3T3 cells exposed to mild-H₂O₂. Thirteen differentially expressed proteins were identified by MS and two of them were further validated by Western blot. The results revealed that exposure to mild-H₂O₂ for 12 h cause up-regulated expression of DJ-1, glutathione S-transferase P 1, DNA ligase I, dynamin 2, nucleophosmin, and down-regulated expression of nucleoside diphosphate kinase A, enolase-α, barrier-to-autointegration factor 1, metastasis associated protein 1, glycosytransferase-like domain containing protein 1, synaptonemal complex protein 1, alpha-centractin, bromodomain, and PHD finger containing 1 (BRPF1). Most of the identified proteins are supported as nuclear proteins localized by previous research. The findings may provide some clues to elucidate cell responses to H₂O₂ and the potential mechanism underlying protection against oxidative stress in fibroblast cells.

Quenched Substrates for Live-Cell Labeling of SNAP-Tagged Fusion Proteins with Improved Fluorescent Background

Recent developments in fluorescence microscopy raise the demands for bright and photostable fluorescent tags for specific and background free labeling in living cells. Aside from fluorescent proteins and other tagging methods, labeling of SNAP-tagged proteins has become available thereby increasing the pool of potentially applicable fluorescent dyes for specific labeling of proteins. Here, we report on novel conjugates of benzylguanine (BG) which are quenched in their fluorescence and become highly fluorescent upon labeling of the SNAP-tag, the commercial variant of the human O(6)-alkylguanosyltransferase (hAGT). We identified four conjugates showing a strong increase, i.e., >10-fold, in fluorescence intensity upon labeling of SNAP-tag in vitro. Moreover, we screened a subset of nine BG-dye conjugates in living Escherichia coli and found them all suited for labeling of the SNAP-tag. Here, quenched BG-dye conjugates yield a higher specificity due to reduced contribution from excess conjugate to the fluorescence signal. We further extended the application of these conjugates by labeling a SNAP-tag fusion of the Tar chemoreceptor in live E. coli cells and the eukaryotic transcription factor STAT5b in NIH 3T3 mouse fibroblast cells. Aside from the labeling efficiency and specificity in living cells, we discuss possible mechanisms that might be responsible for the changes in fluorescence emission upon labeling of the SNAP-tag, as well as problems we encountered with nonspecific labeling with certain conjugates in eukaryotic cells.

Functional Selection of Phagocytosis-Promoting Genes: Cell Sorting–Based Selection

Phagocytosis is a critical host defense mechanism that clears invading pathogens, apoptotic cells, and cell debris; it is an essential process for normal development, tissue remodeling, immune response, and inflammation. Here, a functional selection strategy was used to isolate novel phagocytosis-promoting genes. After the retroviral transfer of mouse brain cDNA library into NIH3T3 mouse fibroblast cells, cell sorting was used to select the cells that phagocytosed fluorescent zymosan particles. The cDNAs were retrieved from the selected cells and identified by DNA sequencing as eIF5A, Meg3, Tubb5, Sparcl-1, Uchl-1, Bsg (CD147), Ube2v1, and Pamr1. The phagocytosis-promoting activity for some of these cDNAs was confirmed by transient transfection in the independent phagocytosis assays. Thus, the unbiased selection procedure successfully identified multiple phagocytosis-promoting genes. The selection method can be applied to other cell-based assays where cells with a desired phenotype can be physically separated. Moreover, the new gene targets uncovered in this study could be relevant to biomolecule screening in search of phagocytosis-regulating agents. In a small-scale screen, a series of imidazopyridine compounds was tested to identify the small molecules that modulate eIF5A-mediated phagocytic activity. Several compounds that influenced the phagocytic activity can be further used as chemical-genetic tools to delineate the mechanisms of eIF5A action and be potential drug candidates that are capable of therapeutically modulating phagocytic activity.

14-3-3γ Induces Oncogenic Transformation by Stimulating MAP Kinase and PI3K Signaling

The 14-3-3 proteins are a set of highly conserved scaffolding proteins that have been implicated in the regulation of a variety of important cellular processes such as the cell cycle, apoptosis and mitogenic signaling. Recent evidence indicates that the expression of some of the family members is elevated in human cancers suggesting that they may play a role in tumorigenesis. In the present study, the oncogenic potential of 14-3-3γ was shown by focus formation and tumor formation in SCID mice using 14-3-3γ transfected NIH3T3 mouse fibroblast cells. In contrast, 14-3-3σ, a putative tumor suppressor, inhibited NIH3T3 transformation by H-ras and c-myc. We also report that activation of both MAP kinase and PI3K signaling pathways are essential for transformation by 14-3-3γ. In addition, we found that 14-3-3γ interacts with phosphatidylinositol 3-kinase (PI3K) and TSC2 proteins indicating that it could stimulate PI3K signaling by acting at two points in the signaling pathway. Overall, our studies establish 14-3-3γ as an oncogene and implicate MAPK and PI3K signaling as important for 14-3-3γ induced transformation.