Minicollect lithium heparin tubes

Blood was extracted using a 1 ml syringe with an 18 G needle from the heart of euthanized mice immediately after death. Blood samples were collected in 1 ml MiniCollect Lithium/Heparin tubes (Greiner-bio), spun down at 400 g for 15 min to separate serum from plasma. Plasma aliquots were added to 1× SDS-PAGE loading buffer/denaturation solution (Thermo Scientific), denatured at 70°C for 10 min and separated on a 3-8% Tris-acetate SDS-PAGE gel (Thermo Scientific). After electrophoresis, gels were washed three times for 5 min with water, stained with Coomassie Blue (SimplyBlue Safe stain, Thermo Scientific) and destained with deionized water for 24 h.

The in vivo experiments were performed in accordance with the Salzburg Animal Care and Use Committee (Study approval no. 20901-TVG/112/6-2018). Animals were maintained under specific pathogen-free conditions and care conformed to the Austria Act on Animal Experimentation. Mice had ad libitum access to water and chow. A total of 1 × 107 cells in 200 µL of a 1:1 mixture of matrigel (Corning, New York, NY, USA) and serum-free medium were subcutaneously injected into the right flank of 5- to 7-week-old female CD-1 nude mice (n = 10–13) (Charles River, Sulzfeld, Germany). Control animals (n = 9) were kept tumor-free. Blood glucose and beta-hydroxybutyrate levels were monitored over time (Precision Xceed System, Abbott, Chicago, IL, USA). For metabolomics analysis, blood was taken by cardiac puncture and collected in MiniCollect^® Lithium Heparin tubes (Greiner Bio-One, Kremsmünster, Austria) and centrifuged for 3 min at 2000 g. Plasma samples were stored at −80 °C until metabolomics analysis. Tumor size at the day of collection is shown in Figure S16. When tumors were harvested, one half of the tumor was snap frozen in liquid nitrogen for metabolomics analysis while the other half was formalin-fixed and paraffin-embedded (FFPE) for histological analysis.

Blood was collected using the submandibular vein method with 21 G needle directly into MiniCollect® Lithium Heparin tubes (Cat # 450537; Greiner). Plasma was obtained by 10 min centrifugation at 2000 rpm. 25 µl were taken for DNA extraction using the Quick-DNA™ MiniPrep kit (Cat #D3024; Zymo Research). The level of the mitochondrial ND2 in the extracted DNA was analyzed by qPCR and was normalized to beta-actin. The primer sequences are shown in Appendix Table S5.