{"doi":"10.3390/ijms26136255","title":"Methods for Mitochondrial DNA Damage and Depletion in Immortalized Trabecular Meshwork Cells","abstract":"Mitochondrial DNA (mtDNA) damage in trabecular meshwork (TM) cells occurs in open-angle glaucoma (OAG). However, current in vitro models for OAG-like changes in TM cells do not explicitly incorporate mtDNA damage. This work validated two methods of mtDNA damage in immortalized TM cells and assessed OAG-associated expression changes. mtDNA was depleted in TM-1 cells via both ethidium bromide (EtBr) treatment and doxycycline (Dox) induction of a mutant (Y147A) version of Uracil DNA Glycosylase 1 (UNG1) in TM-1 cells (TM-1rtTAadv-TRE-UNG1Y147A). Levels of mitochondrial proteins (ATP5F1A, COXII, and COXIV) were measured via western blot. mtDNA levels and mRNA for OAG-associated transcripts (CTGF, FN1, PAI1, and SFRP1) were measured by qPCR. There was a statistically significant decrease in mtDNA levels per cell at all treatment times in both EtBr-treated TM-1 cells and induced TM-1rtTAadv-TRE-UNG1Y147A cells. Protein levels of ATP5F1A were not significantly changed; COXII and COXIV showed significant decreases after both EtBr and Dox induction. Both models resulted in upregulation of CTGF, FN1, and PAI1; additionally, EtBr treatment but not Dox induction resulted in SFRP1 upregulation. In conclusion, two models of mitochondrial depletion were demonstrated in immortalized TM cells; damage was associated with increases in OAG-associated transcripts, supporting a link between mitochondrial damage and glaucoma phenotypes.","journal":"International Journal of Molecular Sciences","year":2025,"id":569485,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9595,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1474688,"name":"Emily Tsaturian","orcid":null,"position":1,"is_corresponding":false},{"id":690400,"name":"Linlin Zhao","orcid":"0000-0002-8821-4198","position":2,"is_corresponding":false},{"id":1228660,"name":"Joshua T. Morgan","orcid":"0000-0001-8033-0285","position":3,"is_corresponding":false},{"id":1474687,"name":"Shane P. Kennedy","orcid":null,"position":0,"is_corresponding":true}],"reference_count":128,"raw_metadata":null,"created_at":"2026-07-19T02:56:59.652443Z","pmid":"40650044","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}