U 13c palmitate

Metabolic flux experiments were performed when cells reached 90% confluence. The medium for ¹³C glucose labeling experiments contained 10 mM (U-¹³C) glucose (Cambridge Isotope Labs, MA, USA) and 2 mM unlabeled glutamine (Sigma-Aldrich, USA). Medium for ¹³C glutamine labeling experiments contained 10 mM unlabeled glucose and 2 mM (U-¹³C) glutamine (Cambridge Isotope Labs). Medium for ¹³C palmitate labelling experiments contained 0.1 mM (U-¹³C) palmitate (Sigma-Aldrich). After treatment, cells were washed thrice with PBS and subsequently treated with pre-cold methanol (80% v/v). At the same time, parallel dishes were used to count cell numbers. Metabolite extractions were then analyzed using LC-MS with a C18 column. Relative metabolite abundances were determined by normalizing the abundances of each metabolite to the internal standard and cell number. The incorporation of ¹³C atoms was denoted as m + n, where n was the number of ¹³C atoms.

At day 9 of differentiation C3H/10T1/2 cells previously infected with Scramble or shHDAC3 adenoviral vectors were treated with 100 μM [U-¹³C]Palmitate (Sigma Aldrich) and 1 mM Carnitine (Sigma Aldrich) for 30 min. [U-¹³C]Palmitate and Carnitine solutions were prepared as described previously³⁷ . Methanolic extracts of cells were analyzed by electrospray ionization flow injection analysis tandem mass spectrometry (FIA-MS/MS). For citrate, acetylCoA, palmitate, and malonylCoA each isotopomer was resolved by m/z ratios and quantified by Mass Distribution Vector (MDV), as described previously^{38 (link)}.
Then, the percentage of MDV for each isotopomer was normalized by protein content, measured by BCA Assay (Euroclone).