Dmem without glucose

The glycolytic function was measured through the Agilent Seahorse XF Glycolysis Stress Test (Agilent Technologies, Santa Clara, CA, USA. In brief, 30,000 CTRL or iLIN28B cells per well were seeded in an XF96 plate left overnight at 37 °C, in a 5% CO₂ incubator in complete medium. The day after, medium was replaced with DMEM without glucose, L-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) enriched with 2 mM of L-glutamine (pH 7.4) and after 1 h of incubation in a 37 °C non-CO₂ incubator, the plate was transferred to the Seahorse XFe96 Extracellular Flux Analyzer to quantify the extracellular acidification rate (ECAR, in [mpH/min]) and the oxygen consumption rate (OCR, in [pmol/min]). The following final concentrations of compounds were used: glucose 10 mM (Sigma-Aldrich, St. Louis, MO, USA), Oligomycin 1 µM (Sigma-Aldrich, St. Louis, MO, USA), and 2-DG 50 mM (TCI EUROPE, Zwijndrecht, Belgium). The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were normalized using total protein amount in each well obtained using BCA assays (Thermo Fisher Scientific, Waltham, MA, USA).

Parental HEK293 cells and FH^dim cells were cultured in DMEM without glucose, glutamine, phenol red, sodium pyruvate and sodium bicarbonate (Sigma-Aldrich Co., LLC.) supplemented with 20 mM glucose, 2 mM glutamine, 44 mM sodium bicarbonate and 10% dialysed FBS (Thermo Fisher Scientific, Inc.), unless otherwise mentioned. Parental and FH^dim cells were seeded into a 60 mm dish at 5.0 × 10⁵ cells/dish and 8.0 × 10⁵ cells/dish, respectively, followed by culture at 37 °C and 5% CO₂ in air. The culture medium was replaced with fresh medium at 16 h after cell seeding (this time point was set as 0 h). Cell counting was performed using a TC20 Automated Cell Counter (Bio-Rad Laboratories, Inc.). Each specific growth rate of both types of cell was determined based on a semi-logarithmic plot of total cell number versus time. For the ¹³C-labelling experiments, the culture medium was replaced with one containing [1,2-¹³C]glucose (99% purity; Cambridge Isotope Laboratories, Inc.) or [U-¹³C]glutamine (98% purity; Sigma-Aldrich Co., LLC).

Cells were cultured in DMEM without glucose, glutamine, phenol red, sodium pyruvate and sodium bicarbonate (Sigma-Aldrich Co., LLC) supplemented with 20 mM glucose, 2 mM glutamine, 44 mM sodium bicarbonate and 10% FBS (HyClone Laboratories, Inc.). Mitochondrial and cytosolic fractionation was performed using a commercially available kit (Cell Fractionation Kit-Standard; Abcam). Briefly, 3.0 × 10⁶ cells were suspended in the attached Buffer A and treated with Detergent I. After incubation at room temperature for 7 min, the sample was centrifuged at 5,000×g and 4 °C for 1 min, after which the supernatant containing the cytosolic fraction was collected. The resulting cell pellet was resuspended in Buffer A and treated with Detergent II. After incubation at room temperature for 10 min, the sample was centrifuged at 5,000×g and 4 °C for 1 min, after which the supernatant containing the mitochondrial fraction was collected. Each mitochondrial and cytosolic fraction sample was aliquoted for western blotting and FH activity assay using Colorimetric Fumarase Activity Assay Kit (Abcam). For FH activity assay, after the sample had been mixed with the attached substrate, enzyme mix and developer solution, absorbance at 450 nm was measured using Versamax (Molecular Devices, LLC) in the kinetic mode at 37 °C for 120 min.

The 8988 T, BxPC3, and MIAPaCa2 human pancreatic cancer cell lines from the American Type Culture Collection (ATCC; Manassas, VA, USA) were kindly provided by Yun-Hee Kim (National Cancer Center, Korea). ATG5 KO MEFs were generated by mating ATG5 heterozygous mice generously provided by Dr. Noboru Mizushima (Tokyo Medical and Dental University, Bunkyo-Ku, Japan)44 (link). All cells were maintained in a 5% CO₂ atmosphere at 37 °C in medium supplemented with 10% FBS (Hyclone), 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies). MEFs, 8988 T, and MIAPaCa2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Life technologies). BxPC3 were cultured in RPMI1640 (Life technologies). For either glucose or glutamine starvation, DMEM without glucose (Sigma-Aldrich) or DMEM without glutamine (Life Technologies) was supplemented with 10% dialyzed-FBS (Life technologies). Constitutively activated Ras GTPase plasmids were from Addgene. pBabe K-Ras G12V (#9052) was generously provided by William Hahn through Addgene. Oncogenic Ras-expressing MEFs were generated following standard protocols for retrovirus transduction.

The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured in cell cultures in DMEM without glucose, L-glutamine, phenol red, sodium pyruvate and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) using the Seahorse XF Cell Mito Stress Test Kit and the Seahorse XFe96 Analyzer (Seahorse Bioscience, North Billerica, MA, USA) under basal conditions and in the presence of the inhibitor of the oxidative phosphorylation oligomycin (1 μM), the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.25 μM for PC3 cells and 0.5 μM for C4-2B cells) and the inhibitors of complex III and I of the respiratory chain antimycin A (0.5 μM) and rotenone (0.5 μM). Basal respiration, ATP-linked respiration, proton leak, maximal respiratory capacity and non-mitochondrial respiration were calculated based on OCR.