Activation of IRE1, PERK and salt-inducible kinases leads to Sec body formation in Drosophila S2 cells.

The phase separation of the non-membrane bound Sec bodies occurs in Drosophila S2 cells by coalescence of components of the endoplasmic reticulum (ER) exit sites under the stress of amino acid starvation. Here, we address which signaling pathways cause Sec body formation and find that two pathways are critical. The first is the activation of the salt-inducible kinases (SIKs; SIK2 and SIK3) by Na+ stress, which, when it is strong, is sufficient. The second is activation of IRE1 and PERK (also known as PEK in flies) downstream of ER stress induced by the absence of amino acids, which needs to be combined with moderate salt stress to induce Sec body formation. SIK, and IRE1 and PERK activation appear to potentiate each other through the stimulation of the unfolded protein response, a key parameter in Sec body formation. This work shows the role of SIKs in phase transition and re-enforces the role of IRE1 and PERK as a metabolic sensor for the level of circulating amino acids and salt. This article has an associated First Person interview with the first author of the paper.

Harnessing changes in open chromatin determined by ATAC-seq to generate insulin-responsive reporter constructs.

BACKGROUND: Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed. RESULTS: We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a weak but significant correlation between chromatin regions annotated to enhancers (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate enhancer regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters. CONCLUSIONS: Insulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression.

Curcumin suppresses cell proliferation and triggers apoptosis in vemurafenib-resistant melanoma cells by downregulating the EGFR signaling pathway.

Melanoma is a malignant tumor with aggressive behavior. Vemurafenib, a BRAF inhibitor, is clinically used in melanoma, but resistance to melanoma cytotoxic therapies is associated with BRAF mutations. Curcumin can effectively inhibit numerous types of cancers. However, there are no reports regarding the correlation between curcumin and vemurafenib-resistant melanoma cells. In this study, vemurafenib-resistant A375.S2 (A375.S2/VR) cells were established, and the functional mechanism of the epidermal growth factor receptor (EGFR), serine-threonine kinase (AKT), and the extracellular signal-regulated kinase (ERK) signaling induced by curcumin was investigated in A375.S2/VR cells in vitro. Our results indicated that A375.S2/VR cells had a higher IC50 concentration of vemurafenib than the parental A375.S2 cells. Moreover, curcumin reduced the viability and confluence of A375.S2/VR cells. Curcumin triggered apoptosis via reactive oxygen species (ROS) production, disruption of mitochondrial membrane potential (ΔΨm), and intrinsic signaling (caspase-9/-3-dependent) pathways in A375.S2/VR cells. Curcumin-induced apoptosis was also mediated by the EGFR signaling pathway. Combination treatment with curcumin and gefitinib (an EGFR inhibitor) synergistically potentiated the inhibitory effect of cell viability in A375.S2/VR cells. The present study provides new insights into the therapy of vemurafenib-resistant melanoma and suggests that curcumin might be an encouraging therapeutic candidate for its drug-resistant treatment.

Ataxin 2-binding protein 1 is a context-specific positive regulator of Notch signaling during neurogenesis in Drosophila melanogaster.

The role of the Notch pathway during the lateral inhibition that underlies binary cell fate choice is extensively studied, but the context specificity that generates diverse outcomes is less well understood. In the peripheral nervous system of Drosophila melanogaster, differential Notch signaling between cells of the proneural cluster orchestrates sensory organ specification. Here we report functional analysis of Drosophila Ataxin 2-binding protein 1 (A2BP1) during this process. Its human ortholog is linked to type 2 spinocerebellar ataxia and other complex neuronal disorders. Downregulation of Drosophila A2BP1 in the proneural cluster increases adult sensory bristle number, whereas its overexpression results in loss of bristles. We show that A2BP1 regulates sensory organ specification by potentiating Notch signaling. Supporting its direct involvement, biochemical analysis shows that A2BP1 is part of the Suppressor of Hairless [Su(H)] complex in the presence and absence of Notch. However, in the absence of Notch signaling, the A2BP1 interacting fraction of Su(H) does not associate with the repressor proteins Groucho and CtBP. We propose a model explaining the requirement of A2BP1 as a positive regulator of context-specific Notch activity.

Identification and characterization of four Drosophila suzukii cellularization genes and their promoters.

BACKGROUND: The spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product. RESULTS: Here, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5' flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells. CONCLUSIONS: The similarities in the gene coding and 5' flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

Spiroplasma eriocheiris Enters Drosophila Schneider 2 Cells and Relies on Clathrin-Mediated Endocytosis and Macropinocytosis.

Spiroplasma eriocheiris causes great economic losses in the crustacean aquaculture industry. However, the mechanism of S. eriocheiris infecting host cells has been poorly studied. We established a Spiroplasma-infected Drosophila Schneider 2 (S2) cell model and investigated its pathogenic mechanism. First, S. eriocheiris induced S2 cell apoptosis and necrosis, seriously decreased cell viability, and increased the production of intracellular reactive oxygen species. Further research showed that S. eriocheiris can invade S2 cells, and the number of copies of intracellular spiroplasmas is sharply increased by 12 h postinfection. In addition, S. eriocheiris can cause S2 cells to form typical inclusion bodies and exhibit large vacuoles. Second, S. eriocheiris is internalized into S2 cells and strongly inhibited through blocking clathrin-mediated endocytosis using chlorpromazine and dynasore. Inhibitors of macropinocytosis, protein kinase C and myosin II, cause a significant reduction in S. eriocheiris in S2 cells. In contrast, disruption of cellular cholesterol by methyl-ß-cyclodextrin and nystatin has no effect on S. eriocheiris infection. These results suggest that the entry of S. eriocheiris into S2 cells relies on clathrin-dependent endocytosis and macropinocytosis, but not via the caveola-mediated endocytic pathway. In addition, the intracellular numbers of S. eriocheiris are dramatically reduced after S2 cells are treated with cytoskeleton-depolymerizing agents, including nocodazole and cytochalasin B. Thus, cellular infection by S. eriocheiris is related to microtubules and actin filaments. This research successfully shows for the first time that S. eriocheiris can invade Drosophila S2 cells and provides a process for S. eriocheiris infection.

Drosophila ML-DmD17-c3 cells respond robustly to Dpp and exhibit complex transcriptional feedback on BMP signaling components.

BACKGROUND: BMP signaling is involved in myriad metazoan developmental processes, and study of this pathway in Drosophila has contributed greatly to our understanding of its molecular and genetic mechanisms. These studies have benefited not only from Drosophila's advanced genetic tools, but from complimentary in vitro culture systems. However, the commonly-used S2 cell line is not intrinsically sensitive to the major BMP ligand Dpp and must therefore be augmented with exogenous pathway components for most experiments. RESULTS: Herein we identify and characterize the responses of Drosophila ML-DmD17-c3 cells, which are sensitive to Dpp stimulation and exhibit characteristic regulation of BMP target genes including Dad and brk. Dpp signaling in ML-DmD17-c3 cells is primarily mediated by the receptors Put and Tkv, with additional contributions from Wit and Sax. Furthermore, we report complex regulatory feedback on core pathway genes in this system. CONCLUSIONS: Native ML-DmD17-c3 cells exhibit robust transcriptional responses to BMP pathway induction. We propose that ML-DmD17-c3 cells are well-suited for future BMP pathway analyses.

A Drosophila genetic screen for suppressors of S6kinase-dependent growth identifies the F-box subunit Archipelago/FBXW7.

This study was designed to identify novel negative regulators of the Drosophila S6kinase (dS6K). S6K is a downstream effector of the growth-regulatory complex mTORC1 (mechanistic-Target-of-Rapamycin complex 1). Nutrients activate mTORC1, which in turn induces the phosphorylation of S6K to promote cell growth, whereas fasting represses mTORC1 activity. Here, we screened 11,000 RNA-interfering (RNAi) lines and retained those that enhanced a dS6K-dependent growth phenotype. Since RNAi induces gene knockdown, enhanced tissue growth supports the idea that the targeted gene acts as a growth suppressor. To validate the resulting candidate genes, we monitored dS6K phosphorylation and protein levels in double-stranded RNAi-treated S2 cells. We identified novel dS6K negative regulators, including gene products implicated in basal cellular functions, suggesting that feedback inputs modulate mTORC1/dS6K signaling. We also identified Archipelago (Ago), the Drosophila homologue of FBXW7, which is an E3-ubiquitin-ligase subunit that loads ubiquitin units onto target substrates for proteasome-mediated degradation. Despite a previous report showing an interaction between Ago/FBXW7 and dS6K in a yeast two-hybrid assay and the presence of an Ago/FBXW7-consensus motif in the dS6K polypeptide, we could not see a direct interaction in immunoprecipitation assay. Nevertheless, we observed that loss-of-ago/fbxw7 in larvae resulted in an increase in dS6K protein levels, but no change in the levels of phosphorylated dS6K or dS6K transcripts, suggesting that Ago/FBXW7 indirectly controls dS6K translation or stability. Through the identification of novel negative regulators of the downstream target, dS6K, our study may help deciphering the underlying mechanisms driving deregulations of mTORC1, which underlies several human diseases.

Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors.

BACKGROUND: S2 cells are one of the most widely used Drosophila melanogaster cell lines. A series of studies has shown that they are particularly suitable for RNAi-based screens aimed at the dissection of cellular pathways, including those controlling cell shape and motility, cell metabolism, and host-pathogen interactions. In addition, RNAi in S2 cells has been successfully used to identify many new mitotic genes that are conserved in the higher eukaryotes, and for the analysis of several aspects of the mitotic process. However, no detailed and complete description of S2 cell mitosis at the ultrastructural level has been done. Here, we provide a detailed characterization of all phases of S2 cell mitosis visualized by transmission electron microscopy (TEM). RESULTS: We analyzed by TEM a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behaviors. This unbiased approach provided a comprehensive ultrastructural view of the dividing cells, and allowed us to discover that S2 cells exhibit a previously uncharacterized behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of endoplasmic reticulum membranes, which associate with mitochondria, presumably to prevent their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. CONCLUSIONS: We discovered several previously unknown features of membrane and MT organization during S2 cell mitosis. The genetic determinants of these mitotic features can now be investigated, for instance by using an RNAi-based approach, which is particularly easy and efficient in S2 cells.

Chrysin inhibit human melanoma A375.S2 cell migration and invasion via affecting MAPK signaling and NF-κB signaling pathway in vitro.

Numerous evidences have shown that chrysin induced cytotoxic effects via induced cell cycle arrest and induction of cell apoptosis in human cancer cell lines, however, no information showed that chrysin inhibited skin cancer cell migration and invasion. In this study, we investigated anti-metastasis mechanisms of chrysin in human melanoma cancer A375.S2 cells in vitro. Under sub-lethal concentrations of chrysin (0, 5, 10, and 15 µM) which inhibits cell mobility, migration and invasion of A375.S2 cells that were assayed by wound healing and Transwell filter. That chrysin inhibited MMP-2 activity in A375.S2 cells was investigated by gelatin zymography assay. Western blotting was used to examine protein expression and results indicated that chrysin inhibited the expression of GRB2, SOS-1, PKC, p-AKT (Thr308), NF-κBp65, and NF-κBp50 at 24 and 48 hours treatment, but only at 10-15 µM of chrysin decreased Ras, PI3K, p-c-Jun, and Snail only at 48 hours treatment and only decrease p-AKT(Ser473) at 24 hours treatment. Furthermore, chrysin (5-15 µM) decreased the expression of uPA, N-cadherin and MMP-1 at 24 and 48 hours treatment but only decreased MMP-2 and VEGF at 48 hours treatment at 10-15 µM and 5-15 µM of chrysin, respectively, however, increased E-cadherin at 5-15 µM treatment. Results of confocal laser microscopy systems indicated that chrysin inhibited expression of NF-κBp65 in A375.S2 cells. Based on these observations, we suggest that chrysin can be used in anti-metastasis of human melanoma cells in the future.

Membrane-bound organelles versus membrane-less compartments and their control of anabolic pathways in Drosophila.

Classically, we think of cell compartmentalization as being achieved by membrane-bound organelles. It has nevertheless emerged that membrane-less assemblies also largely contribute to this compartmentalization. Here, we compare the characteristics of both types of compartmentalization in term of maintenance of functional identities. Furthermore, membrane less-compartments are critical for sustaining developmental and cell biological events as they control major metabolic pathways. We describe two examples related to this issue in Drosophila, the role of P-bodies in the translational control of gurken in the Drosophila oocyte, and the formation of Sec bodies upon amino-acid starvation in Drosophila cells.

The Crystal Structure of the Fifth Scavenger Receptor Cysteine-Rich Domain of Porcine CD163 Reveals an Important Residue Involved in Porcine Reproductive and Respiratory Syndrome Virus Infection.

Porcine reproductive and respiratory syndrome (PRRS) has become an economically critical factor in swine industry since its worldwide spread in the 1990s. Infection by its causative agent, PRRS virus (PRRSV), was proven to be mediated by an indispensable receptor, porcine CD163 (pCD163), and the fifth scavenger receptor cysteine-rich domain (SRCR5) is essential for virus infection. However, the structural details and specific residues of pCD163 SRCR5 involved in infection have not been defined yet. In this study, we prepared recombinant pCD163 SRCR5 in Drosophila melanogaster Schneider 2 (S2) cells and determined its crystal structure at a high resolution of 2.0 Å. This structure includes a markedly long loop region and shows a special electrostatic potential, and these are significantly different from those of other members of the scavenger receptor cysteine-rich superfamily (SRCR-SF). Subsequently, we carried out structure-based mutational studies to identify that the arginine residue at position 561 (Arg561) in the long loop region is important for PRRSV infection. Further, we showed Arg561 probably takes effect on the binding of pCD163 to PRRSV during virus invasion. Altogether the current work provides the first view of the CD163 SRCR domain, expands our knowledge of the invasion mechanism of PRRSV, and supports a molecular basis for prevention and control of the virus. IMPORTANCE: PRRS has caused huge economic losses to pig farming. The syndrome is caused by PRRSV, and PRRSV infection has been shown to be mediated by host cell surface receptors. One of them, pCD163, is especially indispensable, and its SRCR5 domain has been further demonstrated to play a significant role in virus infection. However, its structural details and the residues involved in infection are unknown. In this study, we determined the crystal structure of pCD163 SRCR5 and then carried out site-directed mutational studies based on the crystal structure to elucidate which residue is important. Our work not only provides structural information on the CD163 SRCR domain for the first time but also indicates the molecular mechanism of PRRSV infection and lays a foundation for future applications in prevention and control of PRRS.

Improved process conditions for increasing expression of MHC class II protein from a stable Drosophila S2 cell line.

OBJECTIVES: To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. RESULTS: When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR12xHis) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. CONCLUSIONS: Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.

TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules.

The kinase TOR is found in two complexes, TORC1, which is involved in growth control, and TORC2, whose roles are less well defined. Here, we asked whether TORC2 has a role in sustaining cellular stress. We show that TORC2 inhibition in Drosophila melanogaster leads to a reduced tolerance to heat stress, whereas sensitivity to other stresses is not affected. Accordingly, we show that upon heat stress, both in the animal and Drosophila cultured S2 cells, TORC2 is activated and is required for maintaining the level of its known target, Akt1 (also known as PKB). We show that the phosphorylation of the stress-activated protein kinases is not modulated by TORC2 nor is the heat-induced upregulation of heat-shock proteins. Instead, we show, both in vivo and in cultured cells, that TORC2 is required for the assembly of heat-induced cytoprotective ribonucleoprotein particles, the pro-survival stress granules. These granules are formed in response to protein translation inhibition imposed by heat stress that appears to be less efficient in the absence of TORC2 function. We propose that TORC2 mediates heat resistance in Drosophila by promoting the cell autonomous formation of stress granules.

Mechanical coupling of microtubule-dependent motor teams during peroxisome transport in Drosophila S2 cells.

BACKGROUND: Intracellular transport requires molecular motors that step along cytoskeletal filaments actively dragging cargoes through the crowded cytoplasm. Here, we explore the interplay of the opposed polarity motors kinesin-1 and cytoplasmic dynein during peroxisome transport along microtubules in Drosophila S2 cells. METHODS: We used single particle tracking with nanometer accuracy and millisecond time resolution to extract quantitative information on the bidirectional motion of organelles. The transport performance was studied in cells expressing a slow chimeric plus-end directed motor or the kinesin heavy chain. We also analyzed the influence of peroxisomes membrane fluidity in methyl-ß-ciclodextrin treated cells. The experimental data was also confronted with numerical simulations of two well-established tug of war scenarios. RESULTS AND CONCLUSIONS: The velocity distributions of retrograde and anterograde peroxisomes showed a multimodal pattern suggesting that multiple motor teams drive transport in either direction. The chimeric motors interfered with the performance of anterograde transport and also reduced the speed of the slowest retrograde team. In addition, increasing the fluidity of peroxisomes membrane decreased the speed of the slowest anterograde and retrograde teams. GENERAL SIGNIFICANCE: Our results support the existence of a crosstalk between opposed-polarity motor teams. Moreover, the slowest teams seem to mechanically communicate with each other through the membrane to trigger transport.

[RNA immunoprecipitation technique for Drosophila melanogaster S2 cells].

RNA-binding proteins play an important role in RNA metabolism, especially in mRNA biogenesis and subsequent expression patterns regulation. RNA immunoprecipitation (RIP) is a powerful tool for detecting protein-RNA associations. In this paper, we briefly cover the history of this method for analyzing RNA-protein interactions and reviewing a number of modifications of the RIP technique. We also present an adjusted RIP protocol that was modified for Drosophila S2 cell culture. The use of this protocol allows one to perform the efficient precipitation of RNA-protein complexes and harvest RNA in amounts that are sufficient for its downstream analysis.

Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules.

Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the biological role of some of the proteins binding to the microtubule track is to regulate cargo transport along these tracks.

Efficient expression of functional human coagulation factor IX in stably-transfected Drosophila melanogaster S2 cells; comparison with the mammalian CHO system.

OBJECTIVE: To compare stably-transfected Drosophila melanogaster S2 and mammalian Chinese hamster ovary (CHO) cells for the expression and secretion efficiency of biologically-active human coagulation factor IX (hFIX). RESULT: Selection of an hFIX-expressing cell line derived from stably-transfected S2 cells was performed over 2 weeks, while the same procedure required 2 months for stably-transfected CHO cells. Furthermore, the selected S2 cell line was superior in producing of total hFIX protein (70 % increase), biologically-active hFIX (35 % increase), and specific hFIX activity (20 % increase) relative to the selected CHO cell line. Enrichment for functional, fully γ-carboxylated hFIX species via barium citrate adsorption demonstrated that up to 90 % of the hFIX expressed by S2 cells was γ-carboxylated versus 79 % of CHO-expressed hFIX. Inhibition of N-glycosylation by tunicamycin indicated that N-glycosylation of S2-expressed hFIX had occurred to a similar extent as in the CHO-produced hFIX. CONCLUSION: The Drosophila S2 cell system is an attractive candidate for the efficient production of recombinant hFIX as it has the potential of significantly reducing the cell development time, while producing functional hFIX.

Casticin Inhibits A375.S2 Human Melanoma Cell Migration/Invasion through Downregulating NF-κB and Matrix Metalloproteinase-2 and -1.

Casticin is one of the main components from Fructus Viticis, which is widely used as an anti-inflammatory agent. The mechanism of how casticin affects melanoma cell migration and invasion is still not well known. Here we studied the anti-metastasis effects of casticin on A375.S2 melanoma cells by using a non-lethal concentration. First; we used an adhesion assay to test the A375.S2 cells' adhesion ability after treatment with casticin. We next investigated the cell migration ability after casticin treatment by using a wound healing assay to prove that the migration of A375.S2 cells can be inhibited by casticin and double checked the results using the transwell-migration assay. The suppressive effects on matrix metalloproteinase-2; and -9 (MMP-2; and -9) activities were examined by gelatin zymography. Furthermore, western blotting was used to investigate the protein level changes in A375.S2 cells. We found that p-EGFR; Ras and p-ERK1/2 are decreased by casticin, indicating that casticin can down-regulate the migration and invasion ability of A375.S2 cells via the p-EGFR/Ras/p-ERK pathway. The NF-κB p65 and p-ERK levels in nuclear proteins are also decreased by treatment with casticin. An EMSA assay also discovered that the NF-κB p65 and DNA interaction is decreased. NF-κB p65 protein level was examined by immunofluorescence staining and also decreased. Our findings suggest that casticin has anti-metastatic potential by decreasing the invasiveness of A375.S2 cells. We also found that casticin suppressed A375.S2 cell proliferation and cell adhesion ability, but did not affect cell death, as examined using cytometry and a collagen adhesion assay. Based on these observations, casticin could be used as an inhibitor of migration and invasion of human melanoma cells in the future.

Isolation and characterization of recombinant murine Wnt3a.

Wnt proteins are a family of morphogens that possess potent biological activity. Structure-function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu(2+)-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects.