Figure 2.
Reductive Carboxylation into Malate Supports Glycolysis in Detached Cells
(A) Levels of isotope labeling of glycolytic intermediates of 293T cells cultured in attached or detached conditions in the presence of 13C6-glucose for 4 h. Peak area levels are normalized to cell number.
(B) Western blot analysis demonstrating pyruvate dehydrogenase phosphorylation in 293T attached and detached cells.
(C) The M + 3/M + 4 ratio of malate in 293T and HeLa detached cells after siRNA knockdown of MDH1 and LDHA.
(D) Lactate levels of 293T and HeLa detached cells after siRNA knockdown of MDH1 and LDHA.
(E) A schematic representation of hydrogen-labeling experiment.
(F) Levels of deuterium-labeled malate from 4-2H1-glucose labeling in detached 293T cells following siRNA knockdown of MDH1.
(G) Levels of M + 3 malate excreted from attached and detached 293T and HeLa cells.
(H) A schematic representation of reductive carboxylation into malate supporting glycolysis by regenerating NAD+ in detached cells.
(A, C, D, F, and G) Data are presented as mean ± SD of triplicate cultures of representative experiments.