Use the BM Chemiluminescence Western Blotting kit (Roche) to detect the immunoreactive bands
Use the BM Chemiluminescence Western Blotting kit (Roche) to detect the immunoreactive bands. Cell Proliferation Assay Cell viability and proliferation were assessed using CCK-8 kit (Beyotime Biotechnology). concentrations and hyperpolarization increased after over-activation of AMPK using the specific activator AICAR or suppression of CFTR activity using specific inhibitor GlyH-101. Under these conditions, proliferation of MKN45 cells was inhibited. These results reveal that AMPK controls the dynamic change in Vmem by regulating CFTR and influencing the intracellular Rabbit Polyclonal to Cytochrome P450 7B1 chloride concentration, which in turn influences cell-cycle progression. These findings offer new insights into the mechanisms underlying cell-cycle arrest regulated by AMPK and CFTR. Introduction Gastric cancer is one of the leading causes of cancer-related mortality worldwide. In 2015, over 750,000 people died of gastric cancer1. Despite advances in diagnostic tools and treatments, the prognosis of gastric cancer patients remains particularly poor, with an overall 5 year survival rate of approximately 20%2. Therefore, understanding the regulatory mechanisms that govern cancer cell proliferation, differentiation, migration, and survival is crucial for the development of TP-0903 new, targeted, and more effective therapeutic approaches. Membrane potential (Vmem), a key bioelectric property of non-excitable cells, plays functional roles in cellular processes such as proliferation, differentiation, and migration3. Vmem refers to the voltage gradient across the plasma membrane that results from the discrepancy in ion concentrations between the cytoplasm and the extracellular environment, and it arises from active and passive ion transport through numerous channels in the cell membrane, each of which has a distinct ion selectivity and permeability3C5. Cells are called depolarized when Vmem becomes less negative, and hyperpolarized when the potential becomes more negative3,6. Sodium, potassium, calcium, and chloride are the major ionic gradients across the cell membrane. In contrast to Na+ and Ca2+, most cell membranes are more permeable to potassium and chloride ions7. Based on the voltage gradients and ion distributions across the cell membrane, the inflow of cations such as sodium and calcium and/or the outflow of intracellular chloride anions can induce depolarization7. Chloride channels, the most abundant anion in all organisms, are believed to contribute to Vmem, and to maintain intracellular pH and cell volume8. The chloride current plays important roles in multiple cellular processes, including the cell cycle and proliferation9. Due to the chloride concentration distribution across the plasma membrane, the opening of a passive chloride flux pathway will travel an influx of chloride down its electrochemical gradient7. Cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-gated chloride channel, is indicated in the apical cell TP-0903 membrane of chloride-secreting epithelial cells10. CFTR isn’t just a secretory chloride channel, but also functions as a conductance regulator, coordinating an ensemble of ion fluxes across the cell membrane11,12. A wide variety of membrane transport proteins are modulated by CFTR, including the epithelial sodium channel (ENaC)13, the outwardly rectifying chloride channel14, sodium/hydrogen exchanger15, calcium-activated chloride channels16, aquaporin 9 water channel17, and anion exchanger18. Therefore, CFTR is an important determinant of the fluctuation of Vmem. Vmem levels are tightly related with mitosis, DNA synthesis, and additional events related to cell proliferation. Dividing cells, especially rapidly dividing malignancy cells, are relatively depolarized, TP-0903 whereas non-dividing and quiescent cells, such as terminally differentiated somatic cells, are relatively hyperpolarized3,19,20. Several studies confirm that Vmem modulation can activate or inhibit proliferation inside a predictable way. In 1960s, Clarence D. Cone Jr. 1st reported that sarcoma cells undergo a transient hyperpolarization before entering mitosis, followed by quick depolarization during M phase, suggesting that Vmem varies throughout the cell cycle21. Further, hyperpolarization reversibly blocks DNA synthesis and mitosis. Hyperpolarization to ?75 mV induces a complete mitotic block in Chinese hamster ovary cells, but cell division can be resumed by depolarization to ?10 mV22. Moreover, sustained depolarization can induce DNA synthesis and mitosis in adult neurons, mouse spleen lymphocytes, and muscle mass cells23C25. Growing data suggest that Vmem and ion channels possess practical functions in malignancy progression, therefore showing prognostic value in medical malignancy therapy26,27. In the Xenopus model, depolarization of embryonic cells by manipulating the activity of native glycine receptor chloride channel induces these drastic TP-0903 changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin28. Ivermectin, an antiparasitic agent, induces cell death and delays tumor growth through a mechanism related to chloride-dependent membrane hyperpolarization in leukemia cells29. In addition, Vmem also emerged as regulators of stem cell behavior and developmental TP-0903 processes4,30,31. Vmem hyperpolarization.