Figure 5. AKG dehydrogenase is required for reductive carboxylation.

(A) Abundance of OGDH protein in 143Bcytb cells transfected with a control siRNA (siLUC) or siRNA directed against OGDH (siOGDH).

(B) Relative abundance of succinate in 143Bcytb cells following transient knockdown of siLUC or siOGDH. Data are average and S.D. of three independent cultures. **p<0.005, Student's t-test.

(C) Abundance of m+5 labeled citrate in 143Bcytb cells after transient knockdown of LUC or OGDH cultured with [U-¹³C]glutamine and unlabeled glucose. Abundance of labeled citrate was calculated by multiplying the relative citrate pool size by the percent citrate m+5. Data are the average and S.D. of three independent cultures. *p< 0.05, Student's t-test.

(D) Abundance of OGDH protein in 143Bwt cells transiently transfected with LUC or OGDH.

(E) Ratio of citrate to AKG in 143Bwt cells following knockdown of LUC or OGDH. Data are the average and S.D. of four independent cultures. **p<0.005, Student's t-test.

(F) Mass isotopomer distribution of citrate in 143Bwt cells following transient knockdown of LUC or OGDH and cultured with [U-¹³C]glutamine and unlabeled glucose. Data are the average and S.D. of three independent cultures. **p<0.005, Student's t-test.

(G) Western blot demonstrating expression of fumarate hydratase lacking its mitochondrial targeting sequence and containing a V5 epitope tag (FHΔMTS), in UOK262 cells. EV, empty vector.

(H) Relative fumarate abundance in UOK262 cells containing or lacking FHΔMTS. Data are the average and S.D. of three independent cultures. *p< 0.05, Student's t-test.

(I) Mass isotopomer distribution of citrate in UOK262 cells containing or lacking FHΔMTS. Data are the average and S.D. of three independent cultures. NS, not significant.