Fig. 3: KRAS promotes the lipoylation of DLST and increases the activity of ACO2 in an IscU2 expression-dependent manner.

a–e PaTu-8988t cells transfected with a control siRNA or IscU2 siRNA were cultured with 2 mM U-¹³C-glutamine for 24 h before metabolite extraction. Mass isotopomer distributions of glutamate (a), α-KG (b), fumarate (c), malate (d), and aspartate (e) are shown (means ± SEM, n ≥ 5). f PaTu-8988t cells were transfected with IscU2 siRNA. Immunoblot analyses were performed with the indicated antibodies. g PaTu-8988t cells transfected with a control siRNA, KRAS siRNA or KRAS siRNA with a vector expressing IscU2. Immunoblot analyses were performed with the indicated antibodies. h Mitochondrial ACO2 activity was analyzed by the in-gel activity assay. Immunoblot analyses were performed with the indicated antibodies. i PaTu-8988t cells were transfected with a control siRNA, ACO2 siRNA, or DLST siRNA. α-KG levels were determined and normalized to the numbers of cells (means ± SEM, n = 3). j PaTu-8988t cells transfected with a control siRNA or KRAS siRNA were cultured with 2 mM U-¹³C-glutamine for 24 h before metabolite extraction. Aspartate m + 3 and m + 4 labeling from U-¹³C-glutamine is shown (means ± SEM, n ≥ 7). k PaTu-8988t cells transfected with a control siRNA or IscU2 siRNA were cultured with 2 mM U-¹³C-glutamine for 24 h before metabolite extraction. Aspartate m + 3 and m + 4 labeling from U-¹³C-glutamine are shown (means ± SEM, n = 6). l Schematic model of oxidative and reductive TCA cycling of α-KG produced from U-¹³C-glutamine. Yellow and blue arrows indicate oxidative and reductive metabolism, respectively. Red circles indicate labeled carbons. m OCR of PaTu-8988t cells transfected with a control siRNA, KRAS siRNA or KRAS siRNA with a vector expressing IscU2 (means ± SEM, n = 3). Immunoblot analyses were performed with the indicated antibodies. Statistical significance was determined by unpaired two-tailed Student’s t-test, ns not significant.