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Materials and Methods Mitochondria were isolated from HeLa cells by differential centrifugation as previously described (Dahal et al., 2018; Tadi et al., 2016). Cells were homogenized, mitochondria were purified by sequential centrifugation, lysed in extraction buffer containing protease inhibitors, and clarified by centrifugation. Protein extracts were aliquoted, stored at −80°C, and their purity was confirmed by immunoblotting using mitochondrial and nuclear marker proteins. The optimal concentration of menadione for reactive oxygen species (ROS) induction was determined by DCFDA staining and flow cytometry. HeLa cells were treated with 10–50 μM menadione for 1 h, and 50 μM was selected for all subsequent experiments. Cells were washed before mitochondrial extraction and downstream analyses. For DNA damage experiments, cells were exposed to γ-irradiation using a Cobalt-60 irradiator and incubated for 1 h before harvesting. Microhomology-mediated end joining (MMEJ) assays were performed using synthetic DNA substrates containing 7- or 10-nucleotide microhomologies following established protocols (Sharma et al., 2015). DNA substrates were incubated with mitochondrial or rat testicular extracts under optimized reaction conditions. Repair products were PCR amplified, separated by denaturing polyacrylamide gel electrophoresis, and quantified by phosphorimaging. Selected repair products were cloned into TA vectors and sequenced to determine repair junctions. Plasmids used for homologous recombination (HR) assays were propagated in Escherichia coli and purified by standard alkaline lysis. HR assays were performed using plasmids pTO223 and pTO231 as described previously (Oppliger et al., 1993; Dahal et al., 2018). Recombinant plasmids were recovered by bacterial transformation, recombination frequencies were calculated, and repair products were validated by restriction enzyme digestion. Non-homologous end joining (NHEJ) assays were performed using radiolabeled DNA substrates incubated with cell-free extracts under optimized conditions. Repair products were analyzed by denaturing polyacrylamide gel electrophoresis and phosphorimaging. Protein expression and mitochondrial purity were assessed by SDS-PAGE and immunoblotting using antibodies against Tim44, VDAC, histone H3, and EndoG. For immunofluorescence, HeLa cells treated with menadione were stained with MitoTracker dyes, immunolabeled with specific primary and fluorescent secondary antibodies, counterstained with DAPI, and imaged using confocal microscopy.
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- Indian Institute of Science BangaloreKarnataka, Bengaluru