1 mM). In some experiments, the AMPK inhibitor compound C (6-[4-(2-Piperidin-1-ylethoxy)-phenyl)]-3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine, Calbiochem, La Jolla, CA) was added 30 minutes

before EFV and maintained throughout the 4-hour incubation period. Cells Pifithrin-�� chemical structure were subsequently centrifuged for 5 minutes at 5000 rpm. Forty microliters of the resulting pellet were introduced into a 4-mm ZrO2 rotor fitted with a 50 μL cylindrical insert, and D2O (approximately 10 μL) was added to the sample for field locking purposes. The rotor was then transferred to the NMR probe, which had been cooled at 10°C to minimize sample degradation.17 The entire HR-MAS study was performed at this temperature, having been initiated when the temperature inside the

probe reached the equilibrium condition (approximately 5 minutes). A Bruker Cooling Unit controlled the temperature by cooling the bearing air flowing into the probe. The HR-MAS spectra were recorded on a Bruker Avance 600 spectrometer operating at a frequency of 600.13 MHz and equipped with a 4-mm triple-resonance HR-MAS probe. Samples were spun for 15 minutes at 5000 Hz to keep the rotation sidebands out of the acquisition Regorafenib nmr window, and one-dimensional proton spectra with water pre-saturation were acquired for each sample. Data were processed using the spectrometer software Topspin 1.3 (Bruker Biospin GmbH, Germany). The peak areas were calculated by deconvolution of the region of interest with in-house MATLAB software. Peaks were fitted to a Voight-shape, and calculated areas were normalized with respect to global spectral intensity. Values are mean ± standard error of the mean (SEM) of 3-8 experiments. Statistical analysis was performed by one-way analysis of variance followed by a Newman-Keuls test for unpaired samples (Graph Pad Software V3.02, La Jolla, CA). Significance was *P < 0.05, **P < 0.01, and ***P < 0.001. Figure 1A shows representative learn more traces of respiring Hep3B cells in control conditions and the acute inhibitory effect of EFV (10 and 25 μM) on the rate of O2 consumption after its addition to the gas-tight chambers, illustrated by the slope of the

curve. Figure1B represents the concentration-dependent reduction in O2 consumption produced by EFV (5-100 μM). The maximal inhibitory effect of EFV was obtained with 50 μM and did not differ from that of rotenone 10 μM (31.25% ± 5.55% of control, n = 3, P < 0.001). Doubling the concentration of EFV to 100 μM did not increase the inhibition of respiration. Unless stated otherwise, 15, 25, and 50 μM of EFV were used in all remaining experiments. Incubation for 4 hours did not augment the inhibitory effect of EFV (10 μM), because levels of O2 consumption were similar to those following acute administration of the drug (n = 3, 69.30% ± 3.04% versus 73.45% ± 7.02%, respectively). Respiration of Hep3B cells was restored after removal of EFV from the medium, which suggests that the effects observed were reversible and related to the presence of drug (67.31% ± 8.