αKG-mediated carnitine synthesis drives DNA repair via histone acetylation

Key Points:

• Multiple ovarian and other cancer cell lines were cultured under specific media conditions, with genetic modifications including cyclin E1 and MYC overexpression, and treatments included DNA double-strand break induction and various inhibitors.
• A comprehensive set of shRNAs, antibodies, inhibitors, and metabolites were employed to manipulate and analyze gene expression, protein levels, enzymatic activity, and metabolic pathways, with detailed protocols for metabolite measurement using high-resolution mass spectrometry.
• Functional assays included crystal violet survival assays, Western blotting, immunofluorescence, DNA fiber analysis, and chromatin immunoprecipitation to assess cellular responses to treatments, DNA damage, and histone modifications.
• In vivo mouse xenograft models were used to evaluate tumor growth and treatment responses to inhibitors such as GSK864 and mildronate alone or in combination with chemotherapeutics, with ethical oversight and randomized group allocation.
• Advanced molecular biology techniques such as CRISPR knockout screening targeting αKG-dependent dioxygenases, stable isotope labeling, mass spectrometry for histone modification analysis, and RNA sequencing were utilized to dissect metabolic and epigenetic regulation in cancer cells.