Bay11 7085

Cells were synchronized at G1/S phase transition by double thymidine block, as previously described.^{48 (link)} Subsequently, at the indicated timepoints, cells were collected for FACS and western blot analysis. Briefly, synchronized cells were centrifuged and ressuspended in ice-cold PBS and fixed under gentle vortexing by dropwise addition of an equal volume of ice-cold 80% ethanol (−20 °C), followed by 30 min on ice. Subsequently, samples were stored at 4 °C for at least 18 h, followed by incubation with RNase A solution (10 μg/ml, in PBS) for 30 min at 37 °C. Finally, propidium iodide (25 μg/ml) was added to the samples for 5 min, before flow cytometry analysis. In selected experiments, cells were incubated with ERK5 inhibitor XMD8-92 (#4132, TOCRIS Bioscience, Bristol, UK) or NF-κB inhibitor BAY11-7085 (#sc-202490, from Santa Cruz Biotechnology, Inc.) at 4 μM, or vehicle (DMSO), when cells were subjected to the second thymidine block, and the presence of inhibitors was maintained until the indicated endpoints.

Mice were anesthetized and implanted with a guide cannula (Plastics One) into the third ventricle at the midline coordinates of 0.5 mm posterior to the bregma and 5 mm below the surface of the skull. After at least 1 week of recovery from surgery, 3 μl vehicle (artificial cerebrospinal fluid, aCSF) with or without inhibitors was injected through the cannula into the third ventricle. To determine the role of diabetes-induced hypothalamic inflammation, PDK2, and lactic acid production in feeding behavior and related pathologies, Bay 11-7085 (an inhibitor of I-κB phosphorylation, Santa Cruz Biotechnology, 1 µg), Ki20227 (an inhibitor of CSF1R, Tocris Bioscience, 1 µg), 4-[3-chloro-4-[[(2 R)-3,3,3-trifluoro-2-hydroxy-2-methylpropanoyl]amino]phenyl]sulfonyl-N,N-dimethylbenzamide (AZD7545; a PDK2 inhibitor; 6.4 nM of CSF concentration), and 3-[[3-[(Cyclopropylamino)sulfonyl]-7-(2,4-dimethoxy-5-pyrimidinyl)-4-quinolinyl]amino]-5-(3,5-difluorophenoxy) benzoic acid (GSK2837808A, a small-molecule inhibitor of LDH; 2 µM of CSF concentration) or sodium oxamate (Oxamate, 25 µg) or vehicle (aCSF or 1% (v/v) DMSO) solution were delivered by multiple icv injections to mice. Daily 12-h food intake in the dark phase and body weight were measured at the time of injections.