| Apoptosis is the gene controlled process of programmed cell death (PCD) that may occur in multicellular organisms. When under physiological or pathological conditions, they could initiate internal mechanisms to end their own lives through a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers), in order to maintain a stable internal environment. Phosphatidylinositol3-kinase(PI3K)-Akt signal pathway have an important role in cell proliferation, cell cycle regulation, mitosis, glycogen metabolism, protein synthesis, cell survival and apoptosis, etc. Recent research point out that many virus can regulate cell signal pathways during the infect process, especially those pathways of controlling cell survival, for the sake of virus replication. PI3K-Akt pathway is one of the various pathways which are controlled by virus. C-Jun N-terminal kinase(JNK), also known as stress activated protein kinase, which is a subtype of MAPK in mammalian cells, activated by cytokines and a series of environmental pressure. The role of JNK pathway in apoptosis is complicated and influenced by intracellular environment. JNK pathway is activated in many genres of cell’s apoptosis, to cause the release of cytochrome C and then form apoptosome which lead to Caspase cascade. Nevertheless, by far the understanding of insect cell signaling pathways is limited. The role of signal pathways in the baculovirus infection process is unclear. In order to explore the relationship between baculovirus-induced insect cell apoptosis pathway and PI3K-Akt or JNK signaling pathways, we used the PI3K inhibitor Wortmannin and JNK inhibitor SP600125to treat SL-1cells infected with AfMNPV and determined the effects of these inhibitors on apoptosis induced by AfMNPV in SL-1cells. After cells infected with AfMNPV in the presence of2.5,25,50μM SP600125or0.3,3,30μM Wortmannin respectively, we observed the cells morphology and apoptotic characterization by light microscope, DAPI staining and detected the level of apoptosis in SL-1cell with flowcytometry. The results showed that the inhibitors significantly inhibited apoptosis induced with AfMNPV in SL-1cells, suggesting that PI3K-Akt and JNK signaling pathway can involve in the apoptosis and the two signaling pathways play an important role during apoptosis induced with AfMNPV in SL-1cells.Reseaches found that the apoptosis process can not be shown clearly by general imaging technologies. The development and application of super-resolution imaging technologies enable us to define the accurate localization of biological molecules at nanometer precision and has been hot issues in recent years in imaging field. Photoactivatable fluorescent proteins (PAFPs) are molecules that switch to a new fluorescent state in response to specific light activation, and play vital roles in super-resolution imaging.However, compared to traditional fluorescent proteins such as GFP or RFP, there are only limited PAFPs with modest performance which need to be optimized, making it an important direction to evolve novel fluorescent proteins for super-resolution microscopy to apoptosis observation.Based on the demand and limitation of current used super-resolution microscopy, we developed novel fluorescent proteins named mGeos2which are derived from mEos2. mGeos2is a series of monomeric green reversibly switchable fluorescent proteins (RSFPs) which could be turned off by488-nm laser light and be reactivated by405-nm laser. When compared with mEos2, mGeos2have such superiorities:high photon output per switch, high photostability, a broad range of switching rate, which make them potentially have various applications. The member of this series, mGeos2-VEM/IEM/VEL, exhibits the highest photon budget and localization precision potential among all green RSFPs, which will definitely make it popular in future apoptosis super-resolution imaging if labeled apoptosome proteins like Apaf-1. |
PDF Full Text Request