― 270 ―homeostasis by inhibiting CRH and ACTH secretion through negative feedback. However, chronic stress can disrupt this feedback mechanism, leading to elevated GC levels and dysregulated HPA axis activity, increasing the risk of aging-related diseases. Aging itself alters HPA axis function and is implicated in diseases like Alzheimer’s, Parkinson’s, Huntington’s, depression, and schizophrenia 3). The kidney, vital in aging-related diseases, is closely linked to adrenal gland function. In the African killifish (Nothobranchius furzeri), a model for aging studies due to its short lifespan and human-like aging symptoms, the adrenal gland (interrenal gland) is dispersed within the kidney tissue, yet remains poorly understood. Through this project, we have acquired invaluable findings, including a semi-quantitative method for assessing ectopic DNA in HeLa cells, insights into the role of DDX41 as a sensor for the cytosolic leakage of mitochondrial DNA, and an investigation into age-related changes in the adrenal tissues and kidney of African Turquoise Killifish (N. furzeri).HeLa cells, derived from cervical cancer (ATCC, CCL-2), were cultured in DMEM with 10% FBS and penicillin-streptomycin at 37°C with 5% CO2. Before transfection, cells were plated at 600 cells/µL density. Stealth RNAi siRNA (TFAM: HSS144251, GBA: HSS178140, Thermo Fisher Scientific) in Lipofectamine RNAiMAX silenced target genes at 10 nM RNA concentration. After three days, total cellular RNA was isolated using TRIzol, and cDNA was synthesized with oligo (dT)20 primers. qPCR utilized TB Green Premix Ex Taq II in a Thermal Cycler Dice Real Time System Lite. Statistical analysis employed GraphPad Prism 9.3.1 with two-sided Student’s t-tests, presenting data as means ± standard errors.2. Immunofluorescence Detection of Ectopic Mitochondrial DNA and Image Analysis:HeLa cells were plated at 600 cells/µL density and treated with Stealth RNAi siRNA (Thermo Fisher Scientific) to suppress target genes, achieving 10 nM RNA concentration. After three days, cells underwent immunofluorescence staining using anti-dsDNA antibody for superior mitochondrial DNA detection. Primary antibodies included anti-dsDNA, anti-histone-H2B, and anti-Hsp60. Confocal microscopy analyzed stained cells, with ImageJ software used for image processing. Adjustments were made to Hsp60 and histone H2B signals based on mitochondrial and nuclear DNA levels. Binarization facilitated dsDNA puncta selection and counting, providing detailed analysis of cellular components and interactions.3. Electron Microscopy:One day before transfection, HeLa cells were plated at 600 cells/µL density. Gene silencing was achieved using Stealth RNAi siRNA (Thermo Fisher Scientific) in Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) with a final RNA concentration of 10 nM. Four days post-siRNA knockdown, cells underwent fixation. Fixed with 2% PFA and 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 37°C, samples were then refrigerated at 4°C for 30 min. After overnight fixation at 4°C in 2% glutaraldehyde, samples were washed and post-fixed in 2% osmium tetroxide. Processed through dehydration, resin embedding, sectioning, staining, and imaging under a transmission electron microscope (TEM).Experimental Methods1. Cell Lines, siRNA Treatment, and Quantitative PCR (qPCR) Analysis:
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