Immunocytochemical Staining Assay Research Articles

Effects of LAIR-1 on hepatocellular carcinoma cell proliferation and invasion via PI3K-AKT-mTOR pathway regulation.

Hepatocellular carcinoma (HCC) is one of the common malignant tumors. Although surgical resection is the best treatment for HCC, many patients with HCC are found to have metastases at the time of initial diagnosis and lose the opportunity for radical treatment. Therefore, the study of the invasion and metastasis of HCC has always been the focus of HCC research. This study aimed to assess the influence of LAIR-1 on HCC cell proliferation and invasion and the relevant mechanisms involved in this process. Immunocytochemical staining assay, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) were used to detect the expression of LAIR-1mRNA and protein in healthy human hepatocyte LO2 and the HCC cell lines HepG2, Bel-7402, MHCC97-H, and Huh-7. Then, we evaluated the cell viability, colony formation, and invasion of MHCC97-H and Huh-7 cells in each group by silencing or overexpressing LAIR-1 expression in MHCC97-H and Huh-7 cells, respectively. WB was used to detect the expression levels of PI3K-AKT-mTOR pathway related proteins. Our findings showed that LAIR-1 can inhibit cell viability, colony formation and invasion in vitro. Meanwhile, LAIR-1 significantly downregulated the expression of PI3K, p-AKT and p-mTOR, which were abolished by the PI3K inhibitor, LY294002. Our study revealed that LAIR-1 inhibited cell proliferation and invasion, probably via suppressing the PI3K-AKT-mTOR pathway.

Bilirubin oxidation end product B prevents CoCl2-induced primary cortical neuron apoptosis by promoting cell survival Akt/mTOR/p70S6K signaling pathway

Bilirubin oxidation end products (BOXes) are associated with the late-developing neurological deficits after subarachnoid hemorrhage (SAH) possibly by direct constricting the cerebral arteries, but their specific impacts on neurons especially in the state of hypoxia, a prominent feature during the late stage of SAH, remain unclear. Here, we explored the effects of BOXes on the primary cortical neurons subjected to CoCl2-induced hypoxia by evaluating the morphological and apoptotic changes of neurons. The present study showed that Z-BOX B but not Z-BOX A greatly alleviated CoCl2-induced neuronal cell deterioration and apoptosis. Immunocytochemical staining assay showed Z-BOX B significantly increased neurite length, the numbers of both secondary and tertiary branches, and the protein level of Synaptophysin. Caspase 3/7 apoptosis assay and DAPI staining showed that Z-BOX B markedly reduced primary cortical neurons apoptosis. The expression of cleaved Caspase-3 was suppressed by Z-BOX B treatment, while the expression of Bcl-xL was upregulated. To further discover the mechanism of the neuroprotective effect observed in Z-BOX B, we found Z-BOX B increased the expression of p-mTOR, p-Akt, and p-p70S6K. In general, our results implicated Z-BOX B may prevent CoCl2-induced primary cortical neurons apoptosis by activating sAkt/mTOR/p70S6K signaling pathway. Hence, the present data may provide new insights into the pathophysiological mechanism of delayed neurological dysfunction after SAH and novel targets for treating SAH.

Supramolecular Nanofibers for Encapsulation and In Situ Differentiation of Neural Stem Cells.

Design and fabrication of fibrous materials by natural biological macromolecules in light of biomimetics to achieve spatially cellular arrangements are highly desirable in tissue engineering. Herein, chromatin-inspired supramolecular fibers formed through the interfacial polyelectrolyte complexation (IPC) process by DNA and histone proteins for encapsulation and in situ differentiation of murine brain-derived neural stem cells (NSCs) are reported. High cell viability of encapsulated NSCs demonstrates the excellent biocompatibility of fibers as 3D scaffolds. Moreover, a cell-adhesive peptide (K6 -PEG-RGD) is introduced into fibers by electrostatic interaction to improve NSCs encapsulation efficiency and prevent them from migrating out of fibers for enhanced spatially cellular arrangement. In situ differentiation of NSCs into oligodendrocytes within fibers is revealed by immunocytochemical staining assay. Due to the robust abilities to encapsulate and in situ differentiate NSCs, these chromatin-inspired supramolecular fibers show great potential in neural system-related tissue.

Expression of NS3/NS4A Proteins of Hepatitis C Virus in Huh7 Cells Following Engineering Its Eukaryotic Expression Vector.

Background:Although the development of novel therapeutic regimens to combat hepatitis C virus (HCV) infection have been speeded up with successful results, no efficient vaccines exist yet.Objectives:This study aimed to construct a eukaryotic expression vector encoding nonstructural proteins, NS3/NS4A, of HCV genotype 3a, and evaluate its expression on Huh7 cell surface.Materials and Methods:The NS3/NS4A sequence was isolated from a patient with HCV-3a chronic infection, cloned into intermediate vector pTZ57R/T, and then used for engineering a mammalian expression vector, pDisplay, to direct the respective protein to the secretory pathway and anchor it to the plasma membrane. The expression of the protein in Huh7 cell, which was transiently transfected with the vector using Lipofectamine, was determined by immunocytochemical staining assay with fluorescein isothiocyanate (FITC)-conjugated antibodies to the HA/myc tags located besides the fusion fragment.Results:The results showed that the fragment was successfully amplified and cloned into a eukaryotic expression vector. Sequencing and enzyme digestion analysis confirmed the cloned gene completion and its correct position in the pDisply-NS3/NS4A plasmid. Immunocytochemical staining revealed that the target protein was expressed as a membrane-anchored protein in the Huh7 cells.Conclusions:This study can serve as a fundamental experiment for the construction of a NS3/NS4A eukaryotic expression vector and its expression in mammalian cells. Further research is underway to evaluate the fragment immunogenicity in lab animal models.

Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

Leptin and Interleukin-1β Modulate Neuronal Glutamate Release and Protect Against Glucose-Oxygen-Serum Deprivation

Molecular mechanism underlying leptin-mediated neuronal protection against glucose-oxygen-serum deprivation (GOSD) insult was investigated by focusing on the interactions among leptin, Interleukin-1beta (IL-1beta) and glutamate and their impacts on the growth of neurons under GOSD. The trypan blue dye exclusion assay, 4', 6-diamidino-2-phenylindole (DAPI) assay, cytokine antibody array assay, immunocytochemical staining assay, glutamate determination kit, immunoblocking and chemical blocking strategies were applied to serve the study goal. Results showed that in response to 6 h of GOSD, cortical neurons can secrete significant amounts of leptin and IL-1beta to protect neurons from GOSD-induced cell damage. Serine/threonine kinase Akt (Akt) and extracellular signal-related kinase (ERK) inhibitors significantly reversed leptin-mediated neuroprotection. GOSD-induced IL-1beta was further enhanced by leptin in Akt/ERK-dependent manner. Blockade of endogenous leptin with specific antibodies significantly inhibited GOSD-induced IL-1beta expression and increased glutamate release from GOSD neurons. IL-1 blockade with IL-1 receptor antagonist (IL-1ra) on the other hand, inhibited leptin-mediated neuroprotection and suppression of glutamate release from GOSD neurons. Pre-treating GOSD neurons with leptin and IL-1beta in combined significantly increased their survival but decreased their releases of glutamate. The results indicate that leptin may act through Akt and ERK signaling pathways to protect neurons from GOSD insult; the protection was in part IL-1beta dependent and through which the glutamate release from GOSD neurons was inhibited. Therapeutic values of leptin and IL-1beta were suggested in the treatment of cerebral ischemia at early stage.

Three-Dimensional Scaffolds as a Model System for Neural and Endothelial ‘In Vitro’ Culture

Biomaterials based on the hydrophobic homopolymer poly(ethyl acrylate), PEA, and its copolymers with hydroxyethyl acrylate, p(EA-co-HEA) and methacrylic acid, p(EA-co-MAAc) were prepared as polymeric scaffolds with interconnected pores of 90 microns and tested in vitro as culture substrates and compared for their impact on the differentiation of neural stem cells (NSC) obtained from the subventricular zone (SVZ) of postnatal rats and human endothelial cells (HUVEC). Immunocytochemical staining assay for specific markers show that p(EA-co-MAAc) scaffolds were suitable substrates to promote cell attachment and differentiation of adult NSC and HUVEC cells.

A longitudinal cohort study in calves evaluated for rotavirus infections from 1 to 12 months of age by sequential serological assays

Using an immunocytochemical staining assay involving six different recombinant baculoviruses with each expressing one of the major bovine rotavirus VP7 (G6, G8 and G10) and VP4 (P6[1], P7[5] and P8[11]) serotypes, we analyzed IgG antibody responses to individual proteins in archival serum samples collected from 31 calves monthly from 1 to 12 months of age during 1974-1975 in Higley, Arizona. Seroresponses to VP7 and VP4, as determined by a fourfold or greater antibody response, were not always elicited concurrently following infection: in some calves, (1) seroresponses to VP7 were detected earlier than to VP4 or vice versa; and (2) a subsequent second seroresponse was detected for VP7 or VP4 only. In addition, a second infection was more likely to be caused by different G and/or P types. Analyses of serum samples showed that the most frequent G-P combination was G8P6[1], followed by G8P7[5], G8P8[11] and G6P6[1].

Influence of the substrate's hydrophilicity on the in vitro Schwann cells viability

A series of polymeric biomaterials including poly (methyl acrylate) (PMA), chitosan (CHT), poly(ethyl acrylate) (PEA), poly(hydroxyethyl acrylate) (PHEA), and a series of random copolymers containing ethyl acrylate and hydroxyethyl acrylate monomeric units were tested in vitro as culture substrates and compared for their impact on the proliferation and expansion of Schwann cells (SCs). Immunocytochemical staining assay and scanning electron microscopy techniques were applied to perform a quantitative analysis to determine the correct maintenance of the cultured glial cells on the different biomaterials. The results strongly suggest that cell attachment and proliferation is influenced by the substrate's surface chemistry, and that hydrophobic biomaterials based on PMA, PEA, and the copolymers PEA and PHEA in a narrow composition window are suitable substrates to promote cell attachment and proliferation of SCs in vitro.

Protective actions of Rubus coreanus ethanol extract on collagenous extracellular matrix in ultraviolet-B irradiation-induced human dermal fibroblasts

Solar ultraviolet (UV) irradiation leads to distinct changes in the skin connective tissues by degradation of collagen, which is a major structural component in the extracellular matrix. UV irradiation induces the production of matrix metalloproteinases (MMP) capable of attacking native fibrillar collagen and responsible for inhibiting the construction of collagenous extracellular matrix. In this study, we attempted to investigate the protective actions of Rubus coreanus ethanol extract (RCE) on the MMP production and the consequent procollagen/collagen degradation in UV-B-irradiated human dermal fibroblasts. The analytical data showed that Rubus coreanus ethanol extract was mostly comprised of cyanidin 3-rutinoside. Pre-treatment of fibroblasts with this extract inhibited UV-B-induced production of MMP-1, MMP-8 and MMP-13 in dose-dependent manners. In addition, Western blot analysis and immunocytochemical staining assay revealed that RCE markedly augmented the cellular levels of procollagen/collagen declined in UV-B-exposed dermal fibroblasts. These results demonstrate that RCE blocks UV-B-induced increase of the collagen degradation by inhibiting MMP production. Thus, RCE may act as an agent inhibiting excessive dermal collagen degradation leading to the skin photoaging.

Antimalarial dihydroartemisinin also inhibits angiogenesis

Dihydroartemisinin, a more water-soluble metabolite of artemisinin derivatives, is a safe and most effective antimalarial analog of artemisinin. In the present study, we investigated the antiangiogenic activity of dihydroartemisinin in vitro and in vivo, and investigated dihydroartemisinin-induced apoptosis in human umbilical vein endothelial cells (HUVEC). Dihydroartemisinin markedly reduced VEGF binding to its receptors on the surface of HUVEC. The expression levels of two major VEGF receptors, Flt-1 and KDR/flk-1, on HUVEC were lower following dihydroartemisinin treatment as shown by an immunocytochemical staining assay. The in vivo antiangiogenic activity was evaluated in the model of chicken chorioallantoic membrane (CAM) neovascularization. Dihydroartemisinin significantly inhibited CAM angiogenesis at low concentrations (5-30 nmol/100 microl per egg). We also investigated both qualitatively and quantitatively the induction of HUVEC apoptosis by dihydroartemisinin. A dose-related (5-80 microM) and time-dependent (6-36 h) increase in dihydroartemisinin-induced HUVEC apoptosis was observed by flow cytometry. Our results suggest that the antiangiogenic effect induced by dihydroartemisinin might occur by induction of cellular apoptosis and inhibition of expression of VEGF receptors. These findings and the known low toxicity of dihydroartemisinin indicate that it might be a promising candidate angiogenesis inhibitor.

Quantification of systemic and local immune responses to individual rotavirus proteins during rotavirus infection in mice.

The purpose of the present study was to develop a quantitative assay that could be used to measure the local and systemic immune responses to specific rotavirus proteins following rotavirus infection of adult mice. To measure these responses, we used an immunocytochemical staining assay of Spodoptera frugiperda (Sf-9) cells which were infected with recombinant baculovirus expressing selected rotavirus proteins. The specificity of the assay was documented by using a series of monoclonal antibodies to individual rotavirus proteins. We observed that the assay had high levels of sensitivity and specificity for a series of VP7- and VP4-specific neutralizing monoclonal antibodies which recognized conformation-dependent epitopes on their target proteins. We also studied immunoglobulin G (IgG) immune responses in serum and IgA immune responses in the stools of mice infected with wild-type murine rotavirus strain EHPw. In both sera and stools, the most immunogenic proteins were VP6 and VP4. VP2 was less immunogenic than VP6 or VP4, and the immune responses to VP7, NSP2, and NSP4 were very low in serum and undetectable in stools.

Proliferative capacity of mononuclear cells in the human lung

In pulmonary sarcoidosis, a chronic granulomatous disorder with different stages of activity, the proliferative capacity of the alveolar mononuclear cells is unknown. To get a closer look at this proliferation we combined pulse labeling (by means of tritium thymidine incorporation and autoradiography) with an immunocytochemical staining assay. This assay revealed on one single slide simultaneously blue CD4 + lymphocytes, brown CD8 + lymphocytes and red macrophages. We were able to show that CD4 + as well as CD8 + lymphocytes were radiolabeled only in the active state of the disease. But macrophage proliferation occurred independently of the activity of the disease. In other words with this combination of techniques, it is possible to differentiate, on a single slide, three subsets of mononuclear cells, in combination with their proliferative behavior.