Clark, Courtney2026-09-022026-09-022026-08-262026-08-31http://hdl.handle.net/1993/40049Glioblastoma (GBM) is an aggressive primary brain malignancy with poor prognosis despite surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy. Development of TMZ resistance remains a major therapeutic challenge and has been associated with altered autophagy, mitochondrial function, and sphingolipid metabolism. BCL-2-like protein 13 (BCL2L13), a mitochondria-localized BCL2 family protein, has been implicated in apoptosis, mitochondrial stress responses, autophagy-related processes, and ceramide-associated signalling; however, its role in TMZ-resistant GBM remains poorly understood. This study investigated associations between BCL2L13, autophagy, mitochondrial bioenergetics, ceramide remodeling, and TMZ resistance. A TMZ-resistant (TMZ-R) U251 GBM cell line was established using an 18-week pulsed TMZ selection protocol and compared with TMZ-non-resistant (TMZ-NR) cells. TMZ response, apoptosis, cell-cycle distribution, autophagy markers, and autophagic structures were assessed using viability assays, flow cytometry, immunoblotting, immunocytochemistry, and transmission electron microscopy. Stable BCL2L13 knockdown was generated by lentiviral transduction. Mitochondrial respiration was evaluated using Seahorse XF analysis, and targeted LC-MS/MS lipidomics characterized ceramide and glycosylated ceramide profiles. TMZ-R cells exhibited reduced TMZ sensitivity and apoptosis, impaired late-stage autophagic processing, altered mitochondrial bioenergetics, and increased BCL2L13 expression. BCL2L13 knockdown altered autophagy-marker dynamics in a context-dependent manner but did not restore TMZ-induced apoptosis. Mitochondrial effects of BCL2L13 knockdown were more pronounced in TMZ-NR cells, whereas TMZ-R cells exhibited pre-existing respiratory impairment with minimal additional effects following knockdown. Lipidomic analysis identified TMZ resistance as the dominant source of ceramide-profile separation, with additional BCL2L13-associated remodeling. TMZ-NR cells demonstrated C16:0 and mid-chain ceramide changes consistent with CerS6-associated remodeling, whereas TMZ-R cells exhibited very-long-chain C22–C24 and glycosylated ceramide patterns consistent with CerS2-associated remodeling. Overall, BCL2L13 is associated with context-dependent changes in autophagy-marker dynamics, mitochondrial bioenergetics, and ceramide metabolism. Although BCL2L13 is upregulated in TMZ-R cells, its knockdown does not restore TMZ sensitivity, supporting its role as a modifier of stress-adaptive processes rather than a direct determinant of established TMZ resistance.engGlioblastomaTemozolomide resistanceBCL2L13AutophagyMitochondiral bioenergeticsCeramide metabolism