2DG disrupts Bak-Mcl-1 association, priming cancer cells without lowering protein levels of Mcl-1.

a, The effect of 2DG on cellular ATP levels. The amounts of ATP were measured during HeLa cell culture with 10 mM 2DG in the presence of 25 mM glucose in DMEM supplemented with 10% serum. b, Protein profiles during 2DG incubation of HeLa cells. Samples were taken from HeLa cells treated as in a, and various protein contents of pro- and anti-apoptotic proteins were analyzed by Western blots. In this experiment, we see increases in Bcl-xL for the first hour. When we repeated the experiment twice, Bcl-xL levels remained the same. c, Mcl-1 levels remained unchanged during 2DG-ABT-induced apoptosis. PPC-1 cells were treated with 10 mM 2DG for 6 hours, 1 µM ABT-263 for 3 hours, or 10 mM 2DG for 6 hours of which the last 3 hours were incubated with 1 µM ABT-263. d, Bak and Mcl-1 do not co-precipitate in 2DG treated cells. PPC-1 cells were treated as in c. Bak and Mcl-1 were immunoprecipitated by specific antibodies conjugated to Protein G-Sepharose and the co-precipitated proteins were analyzed by Western blots. Left Upper Panels, Bak co-precipitates, Right Upper Panels, Mcl-1 co-precipitates, and Left Lower Panels, Protein G-Sepharose precipitates. For technical reasons, we could not immunoprecipitate Bcl-xL and analyze its content.