Figure 3.

Some bioenergetic parameters of HEK293 KO cells.A, equal amounts of cells (10⁶) incubated in complete DMEM were added to the chamber of an O₂ electrode, and rates of O₂ consumption (OCR) were measured under basal conditions and after the sequential additions of oligomycin (1 μg/ml), FCCP (1 μM), and antimycin A (2 μM). B, quantification of OCR is shown normalized to the basal OCR of WT cells. The data are the mean + SEM of three independent experiments, each carried out in triplicate: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, two-way ANOVA followed by multiple comparison analysis using Dunnett's test. C, ATP was measured in neutralized perchloric acid (PCA) lysates with a luciferase assay. D, AMP was measured in lysates processed for LC–MS/MS experiments. E and F, the phosphorylation status of AMPK and PDH measured by immunoblotting with phospho-specific antibodies. Values are normalized to the total levels of the enzymes. Statistical evaluation for (C–F) used one-way ANOVA with symbols as in (B). AMPK, AMP-dependent kinase; DMEM, Dulbecco's modified Eagle's medium; FCCP, (carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone); HEK293, human embryonic kidney 293T cell line; OCR, oxygen consumption rate; PDH, pyruvate dehydrogenase.