Immunofluorescence Staining Of Cells Research Articles

Generation and Characterization of Recombinant Antibody-like ADP-Ribose Binding Proteins.

ADP-ribosylation is an enzyme-catalyzed post-translational modification of proteins in which the ADP-ribose (ADPR) moiety of NAD+ is transferred to a specific amino acid in a substrate protein. The biological functions of ADP-ribosylation are numerous and diverse, ranging from normal physiology to pathological conditions. Biochemical and cellular studies of the diverse forms and functions of ADPR require immunological reagents that can be used for detection and enrichment. The lack of a complete set of tools that recognize all forms of ADPR [i.e., mono-, oligo-, and poly(ADP-ribose)] has hampered progress. Herein, we describe the generation and characterization of a set of recombinant antibody-like ADP-ribose binding proteins, in which naturally occurring ADPR binding domains, including macrodomains and WWE domains, have been functionalized by fusion to the Fc region of rabbit immunoglobulin. These reagents, which collectively recognize all forms of ADPR with different specificities, are useful in a broad array of antibody-based assays, such as immunoblotting, immunofluorescent staining of cells, and immunoprecipitation. Observations from these assays suggest that the biology of ADPR is more diverse, rich, and complex than previously thought. The ARBD-Fc fusion proteins described herein will be useful tools for future exploration of the chemistry, biochemistry, and biology of ADP-ribose.

The endonuclease EEPD1 mediates synthetic lethality in RAD52-depleted BRCA1 mutant breast cancer cells

BackgroundProper repair and restart of stressed replication forks requires intact homologous recombination (HR). HR at stressed replication forks can be initiated by the 5′ endonuclease EEPD1, which cleaves the stalled replication fork. Inherited or acquired defects in HR, such as mutations in breast cancer susceptibility protein-1 (BRCA1) or BRCA2, predispose to cancer, including breast and ovarian cancers. In order for these HR-deficient tumor cells to proliferate, they become addicted to a bypass replication fork repair pathway mediated by radiation repair protein 52 (RAD52). Depleting RAD52 can cause synthetic lethality in BRCA1/2 mutant cancers by an unknown molecular mechanism.MethodsWe hypothesized that cleavage of stressed replication forks by EEPD1 generates a fork repair intermediate that is toxic when HR-deficient cells cannot complete repair with the RAD52 bypass pathway. To test this hypothesis, we applied cell survival assays, immunofluorescence staining, DNA fiber and western blot analyses to look at the correlation between cell survival and genome integrity in control, EEPD1, RAD52 and EEPD1/RAD52 co-depletion BRCA1-deficient breast cancer cells.ResultsOur data show that depletion of EEPD1 suppresses synthetic lethality, genome instability, mitotic catastrophe, and hypersensitivity to stress of replication of RAD52-depleted, BRCA1 mutant breast cancer cells. Without HR and the RAD52-dependent backup pathway, the BRCA1 mutant cancer cells depleted of EEPD1 skew to the alternative non-homologous end-joining DNA repair pathway for survival.ConclusionThis study indicates that the mechanism of synthetic lethality in RAD52-depleted BRCA1 mutant cancer cells depends on the endonuclease EEPD1. The data imply that EEPD1 cleavage of stressed replication forks may result in a toxic intermediate when replication fork repair cannot be completed.

Lectin ZG16p inhibits proliferation of human colorectal cancer cells via its carbohydrate-binding sites.

Zymogen granule protein 16 (ZG16p) is a soluble lectin that binds to both mannose and heparin/heparan sulfate. It is highly expressed in the human digestive tract and is secreted into the mucus. In this study, we investigated the effect of ZG16p on the proliferation of human colorectal cancer cells. Overexpression of ZG16p in Caco-2 cells decreased cell growth. Recombinant ZG16p markedly inhibited proliferation of Caco-2, LS174T, HCT116 and HCT15 cells. Caco-2 cell growth was not inhibited by two mutated ZG16p proteins, D151A and M5 (K36A, R37A, R53A, R55A and R79A) lacking mannose- and heparin-binding activities, respectively. Immunofluorescent cell staining revealed that ZG16p-D151A maintained its binding to the Caco-2 cell surface, whereas ZG16p-M5 failed to bind to the cells. These results suggest that ZG16p interacts with the cell surface via basic amino acids substituted in ZG16p-M5 and inhibits Caco-2 cell proliferation via Asp151. In addition, growth of patient-derived colorectal tumor organoids in a 3D intestinal stem cell system was suppressed by ZG16p but not by ZG16p-M5. Taken together, our findings indicate that ZG16p inhibits the growth of colorectal cancer cells via its carbohydrate-binding sites in vitro and ex vivo. In this study, a novel pathway in cancer cell growth regulation through cell surface carbohydrate chains is suggested.

BMP7 mediates the anticancer effect of honokiol by upregulating p53 in HCT116 cells.

Colorectal cancer (CRC) is the second leading cause of cancer death. Hence, there is a great need to explore new efficacious drugs for the treatment of CRC. Honokiol (HNK), a natural product extracted from magnolia bark, processes various biological activities, including anticancer. In this study, we introduced cell viability assay, western blotting, real-time PCR and immunofluorescent staining to determine the anticancer effect of HNK, and the possible mechanism underlying this biological process. We found that HNK can inhibit the proliferation and induce apoptosis in HCT116 cells in a concentration- and time-dependent manner. HNK activates p53 in HCT116 and other colon cancer cells. Exogenous p53 potentiates the anticancer of HNK, while p53 inhibitor decreases this effect of HNK. Moreover, HNK upregulates the expression of bone morphogenetic protein7 (BMP7) in colon cancer cells; Exogenous BMP7 enhances the anticancer activity of HNK and BMP7 specific antibody reduces this effect of HNK. For mechanism, we found that HNK cannot increase the level of Smad1/5/8; Exogenous BMP7 potentiates the HNK-induced activation of p53. On the contrary, BMP7 specific antibody inhibits the HNK-induced activation of p53 in colon cancer cells and partly decreases the total level of p53. Our findings suggested that HNK may be a promising anticancer drug for CRC; activation of p53 plays an important role in the anticancer activity of HNK, which may be initialized partly by the HNK-induced upregulation of BMP7.

Diacylglycerol Kinase alpha is Involved in the Vitamin E-Induced Amelioration of Diabetic Nephropathy in Mice

Diabetic nephropathy (DN) is one of vascular complications of diabetes and is caused by abnormal protein kinase C activation as a result of increased diacylglycerol (DG) production in diabetic hyperglycaemia. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. Therefore, it is expected that the activation of DGK would ameliorate DN. Indeed, it has been reported that vitamin E (VtE) ameliorates DN in rat by activating DGK, and we recently reported that VtE specifically activates DGKα isoform in vitro. However, whether DGKα is involved in the VtE-induced amelioration of DN in vivo remains unknown. Therefore, we investigated the VtE-induced amelioration of DN in wild-type (DGKα+/+) and DGKα–deficient (DGKα−/−) mice in which diabetes was induced by streptozocin. Several symptoms of DN were ameliorated by VtE treatment in the DGKα+/+ mice but not in the DGKα−/− mice. Moreover, transmission electron microscopy of glomeruli and immunofluorescent staining of glomerular epithelial cells (podocytes) indicated that VtE ameliorates podocyte pathology and prevents podocyte loss in the DGKα+/+ mice but not in the DGKα−/− mice. We showed that VtE can ameliorate DN in mice and that DGKα is involved in the VtE-induced amelioration of DN in vivo, suggesting that DGKα is an attractive therapeutic target for DN.

Establishment of a novel three-dimensional primary culture model for hippocampal neurogenesis.

New neurons are generated in the adult hippocampus throughout life and contribute to the functions of learning and memory. Nevertheless, the mechanisms by which disrupted neurogenesis regulates central nervous system (CNS) disorders are not fully understood. Here, we established a novel 3D culture system of hippocampal neurogenesis using air liquid interface (ALI) culture and Matrigel culture from mouse hippocampus tissues. After isolated mouse hippocampus tissue fragments were seeded into ALI wells and cultured in stemness‐stimulated media containing Wnt, EGF, Noggin and R‐spondin for 7 days, small spheres gradually appeared in the tissues. To identify the cell components, immunohistochemical and immunofluorescence staining were performed. Expression of a mature neuronal cell marker, NeuN was observed in the tissues just after seeding. Expression of a neural stem cell marker, Nestin was observed in the tissues at day 7. To differentiate the Nestin‐positive cells, they were passaged into Matrigel. Expression of Nestin but not an immature neuronal cell marker, doublecortin (DCX) was observed in the isolated cells. After 7 days of Matrigel culture, they showed the neurite outgrowth. Expression of Nestin was decreased compared with the one just after passaging, while DCX expression was increased. Western blotting analysis also showed Nestin expression was decreased, while expression of DCX, a neuronal cell marker, Tuj1 and a granule cell marker, Prox‐1 was increased. Here, we establish the 3D culture of hippocampus tissues that might become a novel in vitro tool for monitoring the process of hippocampal neurogenesis. Our model might shed light into the mechanisms of pathogenesis of CNS disorders.

Predictive role of GSTP1-containing exosomes in chemotherapy-resistant breast cancer

Anthracycline/taxane-based chemotherapy regimens are usually used as neoadjuvant chemotherapies to decrease tumour size and prevent metastasis of advanced breast cancer. However, patients have a high risk of developing chemo-resistance during treatment through still unknown mechanisms. Glutathione S-transferase P1 (GSTP1), which belongs to the family of phase II metabolic enzymes, has been reported to function in detoxifying several anti-cancer drugs by conjugating them with glutathione. Previous studies have identified GSTP1 as a predictor of prognosis and chemo-resistance in breast cancer patients, but the mechanisms governing GSTP1-dependent drug resistance are still unclear. We have found that GSTP1 expression is much higher in adriamycin-resistant cells and their corresponding exosomes. The role of GSTP1-containing exosomes in conferring drug resistance was analysed through cell apoptosis and immunofluorescence staining assays. Furthermore, we analysed 42 cases of paired breast cancer tissues collected before and after anthracycline/taxane-based neoadjuvant chemotherapy by immunohistochemistry. Higher GSTP1 expression was shown in the progressive disease (PD)/stable disease (SD) group than in the partial response (PR)/complete response (CR) group both in the samples collected before and after the chemotherapy treatment. Interestingly, GSTP1 partly re-localized from the cell nucleus to the cytoplasm upon treatment, and similar results were obtained for the exosomal marker Tumour susceptibility gene 101 protein (TSG101), which also increased in the cytoplasm after chemotherapy. After analysing the serum exosomes of 30 patients treated with anthracycline/taxane-based neoadjuvant chemotherapy, we discovered that the levels of GSTP1 in exosomes from patients in the PD/SD group were significantly higher than those in the PR/CR group. Here, for the first time, we investigated a novel role for GSTP1-containing exosomes and their capability to transfer drug resistance and evaluated their clinical use in predicting chemo-resistance.

Heat shock exerts anticancer effects on liver cancer via autophagic degradation of aquaporin 5.

Previous studies described that the expression of aquaporin 5 (AQP5) was altered in tumors of various organs. AQP5 is attracting attention as a new cancer therapeutic target. In the present study, heat shock-induced changes in AQP5 expression were evaluated by immunofluorescent staining(IF) and western blotting(WB) of liver cancer cells. AQP5 knockdown experiments or a heat shock treatment were conducted, and their effects on cell volume, proliferation, cell cycle, the activity of apoptosis and migration/invasion were compared. Cycloheximide(CHX) chase experiments and double IF of AQP5 and light chain 3B(LC3B) were performed to investigate the mechanisms underlying changes in AQP5 expression. The results showed that IF and WB revealed decrease in AQP5 expression on cellular membranes and in the cytoplasm of heated cells. AQP5 knockdown and heat shock similarly decreased cell volume, suppressed migration/invasion and proliferation, and induced early apoptosis and partial G0/G1 arrest. CHX chase experiments revealed that heat shock accelerated the degradation of AQP5, which was rescued under CHX and the autophagy inhibitor, bafilomycinA1 (BafA1). Double IF showed the co-localization of AQP5 and LC3B on BafA1-treated heated cells. In conclusion, we demonstrated that heat shock decreased AQP5 on cellular membranes and in the cytoplasm by activating autophagic degradation, and heat shock and AQP5 knockdown exerted similar anticancer effects, suggesting that heat shock exerts anticancer effects via the autophagic degradation of AQP5.

Effect of Micro/Nano-Patterned Surfaces on Cell Adhesion of Ca9-22 cells

Surface topography influences cell growth and differentiation. In this study, we used a nano-imprinting method to develop a titanium sheet with 500 nm-, 1 μm-, and 2 μm-wide grooved and pillared structures. We investigated the effects of the surfaces with the micro- and nano-structures on Ca9-22 cell adhesion and proliferation. Ca9-22 cells were cultured in DMEM containing 10% fetal bovine serum and counted adhered cells at 1 and 24 h postculture. Scanning electron microscopy was used to assess cell morphology. Immunofluorescence cell staining was used to evaluate vinculin formation to observe the presence of focal contacts at 24 h. There was no difference in cell adhesion between cells cultured on a plane or groove after 1 h. However, at 24 h, the adhesion of cells cultured on the groove was reduced. In addition, the cell adhesion count on the pillar was less than that of cells cultured on a plane at both 1 hour and 24 h post-seeding. Furthermore, in the groove of the Ti sheet after 1 and 24 h, cell expansion occurred in the grooved direction. These results demonstrate that the micro and nano-grooved and pillared structures on the titanium sheet control Ca9-22 cell adhesion and orientation. [DOI: 10.1380/ejssnt.2017.1]

MALAT1 Modulates TGF-β1-Induced Endothelial-to-Mesenchymal Transition through Downregulation of miR-145

Background/Aims: Endothelial-to-mesenchymal transition (EndMT) plays significant roles under various pathological conditions including cardiovascular diseases, fibrosis, and cancer. EndMT of endothelial progenitor cells (EPCs) contributes to neointimal hyperplasia following cell therapy Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that promotes metastasis and cancer. MicroRNA-145 (miR-145) is a tumor suppressor that has been reported to inhibit SMAD3-mediated epithelial-to-mesenchymal transition (EMT) of cancer cells. In the present study, we investigated the role of MALAT1 and miR-145 in EndMT of human circulating EPCs induced by transforming growth factor beta1 (TGF-β1). Methods: Human circulating EPCs were isolated and characterized by fluorescence-activated cell sorting (FACS). Expression levels of EndMT markers were assessed by qRT-PCR and western blotting. Alpha-smooth muscle actin (α-SMA) expression was measured by cell immunofluorescence staining. The regulatory relationship between MALAT1 and miR-145 and its target genes, TGFBR2 (TGFβ receptortype II) and SMAD3 (mothers against decapentaplegic homolog 3) was analyzed using the luciferase reporter assay. Results: We found that EndMT of EPCs induced by TGF-β1 is accompanied by increased MALAT1 expression and decreased miR-145 expression, and MALAT1 and miR-145 directly bind and reciprocally repress each other in these cells. Dual-Luciferase Reporter assay indicated that miR-145 inhibits TGF-β1-induced EndMT by directly targeting TGFBR2 and SMAD3. Conclusions: MALAT1 modulates TGF-β1-induced EndMT of EPCs through regulation of TGFBR2 and SMAD3 via miR-145. Thus, the MALAT1-miR-145-TGFBR2/SMAD3 signaling pathway plays a key role in TGF-β1-induced EndMT.

A simplified procedure for antibody engineering by yeast surface display: Coupling display levels and target binding by ribosomal skipping.

Yeast surface display is a valuable, widely used method for protein engineering. However, current yeast display applications rely on the staining of epitope tags in order to verify full-length presentation of the protein of interest on the cell surface. We aimed at developing a modified yeast display approach that relies on ribosomal skipping, thereby enabling the translation of two proteins from one open reading frame and, in that manner, generating an intracellular fluorescence signal. This improved setup is based on a 2A sequence that is encoded between the protein to be displayed and a gene for green fluorescent protein (GFP). The intracellular GFP fluorescence signal of yeast cells correlates with full-length protein presentation and omits the need for the immunofluorescence detection of epitope tags. For method validation, shark-derived IgNAR variable domains (vNAR) were subjected to affinity maturation using the 2A-GFP system. Yeast library screening of full-length vNAR variants which were detected via GFP expression yielded the same high-affinity binder that had previously been isolated by our group using the conventional epitope tag-based display format. The presented method obviates the need for additional immunofluorescence cell staining, offering an easy and cost-friendly alternative to conventional epitope tag detections.

CCND1 and CCND2 Mutations Are Frequent in Adults with Core-Binding Factor Acute Myeloid Leukemia (CBF-AML) with t(8;21)(q22;q22)

CBF-AML is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a "second hit" to initiate leukemogenesis. Although compared with other AML subtypes patients with CBF-AML have a relatively favorable prognosis, still almost 40% of CBF-AML patients experience relapses of their disease.Mutation assessment of 84 leukemia- and/or cancer-associated genes using a customized targeted next-generation sequencing (NGS) approach was done on samples from 177 adults with CBF-AML [t(8;21), n=68; inv(16)/t(16;16), n=109]. We identified frequent mutations in the CCND1 and CCND2 genes as novel molecular alterations in AML with t(8;21). They were detected in 10 (15%) patients with t(8;21), making CCND1/2 mutations the third most frequently detected mutations in t(8;21) AML patients. In contrast, we found a single CCND2 mutation in only 1 (0.9%) patient with inv(16), and CCND1/2 mutations in only 11 of 1,426 non-CBF-AML patients (0.77%). Testing for CCND1 and CCND2 mutations using Sanger sequencing in additional 25 CBF-AML patients with t(8;21), for whom NGS analysis was not possible, identified 3 CCND1/2 mutated patients, thereby confirming the CCND1/2 mutation frequency in t(8;21) AML (15% in the NGS set, and 12% in Sanger sequencing set). All CCND2 mutations cluster around the highly conserved amino acid residue threonine 280 (Thr280), and encode a degradation-resistant CCND2 protein. Curiously, de novo germline CCND2 mutations affecting the identical amino acid residues Thr280 or Pro281 have been recently found to cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH) type 3, an extremely rare neurodegenerative disorder (Mirzaa et al., Nat Genet. 2014;46:510-5).The variant allele fractions of the detected CCND1/2 mutations were variable, suggesting that these mutations can represent both early and later mutational events. Interestingly, three of the 11 CCND1/2 mutated patients had two different CCND1/2 mutations, suggesting a dependence of their leukemia on this mutational event.The evaluation of mutations in other genes found in CBF-AML patients with CCND1/2 mutations did not reveal any statistically significant co-occurring or mutually exclusive mutations. The most frequent mutation co-occurring with CCND1/2 was ASXL2, detected in 4 of the 10 (40%) cases with t(8;21). Patients with CCND1/2 mutations presented with lower percentages of blood (P=0.02) and bone marrow (P=0.05) blasts than patients without these mutations, but did not differ significantly with regard to other pretreatment characteristics. No differences in the achievement of complete remission, disease-free or overall survival were observed between CCND1/2 mutated and CCND1/2 wild-type patients. Mechanistically, we show that Thr280Ala mutated CCND2 significantly increased cell growth (as assessed by TiterGlo assays and cell counts) in all experimental set-ups. Western blotting revealed an increase of retinoblastoma gene phosphorylation, indicating increased downstream signaling. Moreover, both propidium iodide mediated cell cycle analysis and immunofluorescence staining for phospho-histone H3 indicated an increased mitotic rate and a decreased G1 fraction.In summary, recurrent mutations in CCND1/2 are consistent with the currently known mutational landscape of t(8;21) AML, which is dominated by receptor tyrosine kinase, RAS, and chromatin remodeling gene mutations─all of which can result in targeting of CCND1/2. While CCND1 and CCND2 play a crucial role in the regulation of hematopoietic differentiation and cell proliferation, and their differential activation has been implicated in leukemogenesis, frequent mutations in these genes have not previously been reported in AML. The fact that no outcome difference was seen when comparing CCND1/2-mutated versus wild-type patients may be due to the relatively small sample size, or may possibly reflect the common use of cell cycle affecting high-dose cytarabine-based therapy in CBF-AML patients. Thus, the identification of CCND1/2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease. DisclosuresStone:Merck: Consultancy; Karyopharm: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy; Celator: Consultancy; Agios: Consultancy; Amgen: Consultancy; Novartis: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Tetrandrine inhibits glioma stem-like cells by repressing β-catenin expression.

Cancer stem cells (CSCs) in glioma are often responsible for relapse and resistance to therapy. The purpose of the present study was to confirm the self-renewal and migration inhibitory effects of tetrandrine (Tet), which is a compound extracted from the dried root of Stephania tetrandra S. Moore, toward glioma stem-like cells (GSLCs) and to examine the associated molecular mechanisms. Using a neurosphere culture technique, we enriched the GSLC population from the human glioblastoma cell lines U87 and U251. Cells were analyzed using cell counting kit-8 (CCK-8), western blotting, flow cytometry, transwell assay and immunofluorescence staining. GSLCs displayed properties of neural stem cells, including elevated expression of the cancer stem cell marker ALDH1 and β-catenin. We found that Tet treatment decreased sphere formation in GSLCs in a dose-dependent manner using tumor spheroid formation assay. The GSK3β inhibitor BIO maintained sphere formation and migration capacity in GSLCs, whereas the β-catenin/TCF transcription inhibitor ICG-001 decreased sphere formation and the migration capacity of GSLCs. The proportion of apoptotic GSLCs also increased in response to ICG-001 treatment. These results indicate that β-catenin activity is vital in maintaining neural stem cell traits of GSLCs. Tet inhibits cell viability, neurosphere formation and migration of GSLCs invitro. Importantly, Tet treatment significantly repressed the nuclear translocation and expression of β-catenin and induced apoptosis in GSLCs, as indicated in part by the upregulation of Bax, the cleavage of PARP and the downregulation of Bcl-2. The present study demonstrates that the inhibition of β-catenin in CSCs by Tet could be an effective strategy for the treatment of glioma.

Liraglutide suppresses obesity and induces brown fat-like phenotype via Soluble Guanylyl Cyclase mediated pathway in vivo and in vitro.

Strategies for driving white adipose tissue (WAT) to acquire brown-like characteristics are a promising approach to reduce obesity. Liraglutide has been reported to active brown adipose tissue (BAT) thermogenesis and WAT browning by rapid intracerebroventricular injection in mice. In this study, we investigated the effects and possible mechanisms of liraglutide on WAT browning by chronic treatment. Here, we show that liraglutide significantly decreases body weight of mice and reduces the size of white adipocytes. By quantity polymerase chain reaction, immunoblotting analysis, cell immunofluorescence or immunocytochemical staining, we found liraglutide induced WAT browning because it up-regulated lipolytic activity, BAT, as well as mitochondrial marker genes in inguinal and peripheral renal WAT. We also confirmed liraglutide induced browning of 3T3-L1 because it enhanced expression of BAT and mitochondrial specific genes. In further, we observed that, soluble guanylyl cyclase (sGC) and protein kinase G I (PKGI) were up-regulated by liraglutide in vivo and in vitro; stimulation of sGC elevated expression of BAT markers and PKGI, which suggested that liraglutide induced WAT browning via sGC-dependent pathway. Taken together, this study expands our knowledge on the mechanism of liraglutide inducing WAT browning, and provides a theoretical support for clinical usage of liraglutide on obesity treatment.

A Versatile Method for Immunofluorescent Staining of Cells Cultured on Permeable Membrane Inserts.

BackgroundObtaining high-quality images of cellular structures via immunofluorescence staining is critical for cellular localization studies. Often, these studies cannot be performed in parallel with certain oncology, virology, pharmacokinetic, and drug absorption studies due to model system technicalities requiring the cells to be cultured on porous membranes rather than glass or plastic.Material/MethodsHere, we report a method of immunofluorescent staining of cells cultured on permeable membranes.ResultsAs proof of principle, HeLa cells grown on Transwell® membrane supports were stained with fluorescently labeled antibodies using this modified immunofluorescence staining method and visualized by fluorescent microscopy.ConclusionsThis protocol is a convenient alternative to staining cells on glass coverslips, thereby expanding the scope and applications of this important research tool.

Loading-Induced Heat-Shock Response in Bovine Intervertebral Disc Organ Culture.

Mechanical loading has been shown to affect cell viability and matrix maintenance in the intervertebral disc (IVD) but there is no investigation on how cells survive mechanical stress and whether the IVD cells perceive mechanical loading as stress and respond by expression of heat shock proteins. This study investigates the stress response in the IVD in response to compressive loading. Bovine caudal disc organ culture was used to study the effect of physiological range static loading and dynamic loading. Cell activity, gene expression and immunofluorescence staining were used to analyze the cell response. Cell activity and cytoskeleton of the cells did not change significantly after loading. In gene expression analysis, significant up-regulation of heat shock protein-70 (HSP70) was observed in nucleus pulposus after two hours of loading. However, the expression of the matrix remodeling genes did not change significantly after loading. Similarly, expressions of stress response and matrix remodeling genes changed with application and removal of the dynamic loading. The results suggest that stress response was induced by physiological range loading without significantly changing cell activity and upregulating matrix remodeling. This study provides direct evidence on loading induced stress response in IVD cells and contributes to our understanding in the mechanoregulation of intervertebral disc cells.

LGP2 of black carp plays an important role in the innate immune response against SVCV and GCRV

RIG-I like receptors (RLRs) detect cytosolic RNA virus components and initiate antiviral innate immune response through downstream signaling in vertebrates. In this paper, LGP2 of black carp (Mylopharyngodon piceus) has been cloned and characterized, which is a key member of RLR family. The full-length cDNA of black carp LGP2 (bcLGP2) comprises 2941 nucleotides and the predicted bcLGP2 protein contains 682 amino acids. bcLGP2 shares core homologous structural domains of RLRs, including a N-terminnal DExD/H helicase domain, a helicase superfamily c-terminal domain, and a C-terminal regulatory domain (CTD). bcLGP2 mRNA was constitutively detected in all selected tissues including heart, liver, spleen, kidney, intestine, muscle, skin, gill; and bcLGP2 mRNA level was increased in all the tissues except gill in response to GCRV or SVCV infection. Q-PCR of Mylopharyngodon piceus fin (MPF) cells demonstrated that bcLGP2 transcription was up-regulated by Poly (I:C) treatment, GCRV or SVCV infection, but not by LPS or PMA treatment. Western blot analysis demonstrated that the molecular weight of bcLGP2 was around 80 KDa; and the immunofluorescence staining of both HeLa cells and EPC cells showed that bcLGP2 was a cytosolic protein. EPC cells transfected with plasmid expressing bcLGP2 showed obviously improved antiviral ability against SVCV and GCRV. In general, these data support the conclusion that bcLGP2 functions importantly in the host antiviral innate immune response.

Fabricating small-scale, curved, polymeric structures for biological applications using a combination of photocurable/thermocurable polydimethylsiloxane and phase interactions

This paper describes an easy-to-handle technique for creating curved, millimetrically scaled polymeric structures in order to develop in vitro cell culture devices for biologically relevant applications. For the master mold in this study, the authors used UV-activated photocurable polydimethylsiloxane (PDMS). This product can readily be used to create millimetrically scaled pattern molds by controlling droplet contact angles during deposition onto a flexible paper-based substrate that has been prepatterned with ink/wax. Resultant desired patterns can be transferred onto thermocurable PDMS as arrays of wells for biological applications. By combining photocurable and thermocurable PDMS manufacturing processes, this approach endows PDMS-based structures with unique controllability in terms of size, pattern, and curvature. Providing such features enhances the biocompatibility and practicality of devices so manufactured in that they mimic the natural topography of the extracellular matrix. Additionally, three-dimensional cell culturing and immunofluorescent staining can be demonstrated on this biomimetic platform. This manufacturing method takes only several minutes to complete and does not require complicated facilities in order to fabricate PDMS-based biomedical devices. We believe that this method would be very useful for rapid, economical fabrication of cell-focused assay platforms, which would be particularly useful in resource-limited settings.

PM 7/60 (2) Pantoea stewartii subsp. stewartii

Specific scopeThis Standard describes a diagnostic protocol for Pantoea stewartii subsp. stewartii.Specific approval and amendmentApproved in 2005‐09.Revised in 2016‐04.

Abstract 1687: KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A

Abstract Background & Purpose In recent, KIF5B-RET gene rearrangement has been discovered as a driver oncogene in non-small cell lung cancers. This fusion gene was demonstrated to be able to induce tumorigenesis in vivo mouse model. Hereby, we investigated how KIF5B-RET fusion gene affect tumor differentiation and which transcription factors and signaling pathway are activated. Methods We analyzed cell proliferative activity in transformed HEK293T cells with KIF5B-RET fusion gene (K4) and empty vector (V5) using CCK8 assay. We used light microscopy and H&E staining to observe morphological changes of transformed cells. We evaluated protein and mRNA expressions of four EMT markers (E-cadherin, N-cadherin, Snail, and Twist) and two transcription factors (FOXA2, p-STAT5A) using transcription factor PCR array, Western blot, RT-PCR, immunofluorescence staining. Results K4 cells showed decreased cell proliferative activity compared with V5 cells. V5 cells had spreading pattern alike mesenchymal phenotype when their density was lower. However, their morphology changed to epithelial phenotype as they were overgrown with higher density. On the other hands, K4 cells exhibited the island growing pattern similar to epithelial phenotype in lower density of tumor cells, but changed to mesenchymal phenotype as the density of tumor cells gradually increased. On H&E staining, the cytoplasm of K4 cells was much smaller than that of V5 cells. We observed that E-cadherin mRNA was down-regulated, but N-cadherin and Twist were up-regulated in K4 cells. Based on the results of repeated screening assay, we finally selected two transcription factors, FOXA2 and STAT5A, which were significantly overexpressed in K4 cells. In K4 cells, phosphorylated-STAT5A was increased and FOXA2 was decreased compared to V5 cells. In addition, the expression of E-cadherin was reduced, but p-GSK3β, p-AKT, and p-ERK were markedly increased in K4 cells. The pattern of immunofluorescence staining of V5 and K4 cells were similar to the protein expressions of E-cadherin and p-STAT5A in Western blot analysis. Conclusion Taken together, FOXA2 and E-cadherin are down-regulated and p-STAT5A is up-regulated in HEK-293T cells stably expressing the KIF5B-RET. Our data suggest that KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A. Citation Format: Min-Young Kim, Jung-Young Shin, Jeong-Oh Kim, Kyoung-Hwa Son, Jin Hyoung Kang. KIF5B-RET fusion gene may induce EMT via the regulation of two transcription factors, FOXA2 and STAT5A. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1687.