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. Author manuscript; available in PMC: 2016 Jan 16.
Published in final edited form as: Nat Commun. 2015 Jul 16;6:7530. doi: 10.1038/ncomms8530

Figure 2. 14-3-3σ inhibits cancer metabolic reprogramming.

Figure 2

(a) The broad suppressive impact of 14-3-3σ on cancer metabolism. Nuclear Magnetic Resonance was used to measure the concentrations of important metabolites in HCT116 14-3-3σ+/+ (WT), HCT116 14-3-3σ−/−.

(b) 14-3-3σ decreased the extracellular acidification rate (ECAR) of HCT116 in vitro. ECARs were measured by a Seahorse Extracellular Efflux Analyzer XF96 (XF96). Average±standard deviation (SD), n=3, p<0.05.

(c) 14-3-3σ reduced the extracellular acidification rate (ECAR) of MDA-MB-231 in vitro. ECARs were measured by XF96. Average±SD, n=3, p<0.05.

(d) 14-3-3σ inhibited the oxygen consumption rate (OCR) of HCT116 in vitro. OCRs were measured by XF96. Average±standard deviation (SD), n=3, Student t-test,* P< 0.05.

(e) 14-3-3σ diminished the oxygen consumption rate of MDA-MB-231 in vitro. OCRs were measured by XF96. Average±SD, n=3, Student t-test,* P< 0.05

(f) 14-3-3σ inhibited cancer glutaminolysis as measured by ammonia production assays. Cellular lysate of MDA-MB-231 TetR 14-3-3σ cells were collected to measure ammonia production rate using an ammonia production kit (Sigma Aldrich). Average±SD, n=3, Student t-test,* P< 0.05.

(g) 14-3-3σ decreased cancer mitochondrial mass of MDA-MB-231 in vitro. MDA-MB-231 TetR 14-3-3σ cells were stained with Mitochondria Tracker Green FM (Molecular Probes, Invitrogen), which stains functional mitochondria. Mitochondrial Tracker Green FM signals were analyzed using a BD Biosciences FACS Canto flow cytometer. FlowJo X software was used to build Mitochondrial Tracker Green FM histograms. Non-induced MDA-MB-231 TetR 14-3-3σ cells were used as a control.

(h) Induction of 14-3-3σ expression decreased ATP concentrations in the colorectal cancer cell line HCT116 and the triple negative breast cancer cell line MDA-MB-231 in vitro. HCT116 14-3-3σ−/− TetR 14-3-3σ and MDA-MB-231 TetR 14-3-3σ cancer cells were grown in doxycycline-containing medium (5 ng/ml) for inducing Flag-14-3-3σ expression for 3 days. Non-induced cells were used as a control. Cell lysates from HCT116 14-3-3σ−/− TetR 14-3-3σ cells and MDA-MB-231 TetR 14-3-3σ cells were collected for measuring ATP concentration using an ATP Bioluminescence CLSII kit (Roche) and a luminometer (Biotek). Average±95%CI, n=3, ANOVA,* P< 0.05.