Mak039

Lactate (MAK064, Sigma-Aldrich), LDH (MAK066, Sigma-Aldrich), pyruvate (MAK071, Sigma-Aldrich), and Acetyl-CoA (MAK039, Sigma-Aldrich) concentrations and PDH activity (Abcam) in media and in cells were measured using commercial kits following the manufacturer’s instructions.

Two groups of knockout cells and two groups of wild cells were cultured at the same time, with three replicates in each group. When the cells reached 70% confluence, one group of knockout cells and one group of wild cells were randomly treated with 100 μM acetyl-CoA salt (32140-51-5, Sigma, St. Louis, MO, USA, ≥93%) addition. After 48 h, the cells were collected, acetyl-CoA in the cells was extracted by using an acetyl-coenzyme assay kit (MAK039, Sigma, St. Louis, MO, USA) according to the manufacturer’s instructions. Then, the fluorescence intensity was measured (λ_ex = 535/λ_em = 587 nm) by a Biotek Microplate Reader (Winooski, VT, USA).

Acetyl-CoA (MAK039, Sigma) and H₂O₂ (BC3595, Solarbio) were measured according to the instructions provided by the manufacturers. AICAR Tfase activity assays were performed as described [63 (link), 64 (link)]. Cells were homogenized in 20 mM HEPES-KOH buffer, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM sodium EDTA buffer, 1 mM sodium EGTA buffer, and 1 mM dithiothreitol in the presence of 250 mM sucrose and protease inhibitor cocktail (Roche Diagnostics). Each reaction mixture contained a final concentration of 66 mM Tris-Cl, pH 7.4, 100 mM 10-f-FH4, 50 mM AICAR, and 50 mM KCl. AICAR TFase was assayed using AICAR and 10-f-H2F by following the production of H2F at 298 nm.

Acetyl-CoA was quantified in a microcuvette format by a spectrophotometric assay using a sequential, coupled enzymatic reaction as described earlier [24 ] with minor modifications. Mainly, the assay was downscaled to a final volume of 0.503 mL (instead of 2.01 mL) to reduce consumption of metabolite extract. Furthermore, for a most exact determination, the amount of acetyl-CoA in the assay should be kept below 50 nmol. Alternatively, acetyl-CoA was quantified in a 96-well microplate format by a commercial fluorescence assay kit (MAK039, Sigma Aldrich, USA) according to the supplier’s instructions. Deviating from this protocol, the entire fluorescence kinetics at Ex/Em = 535/587 nm was recorded for up to 30 min for retroactive choice of the endpoint.

The PHB synthase activity was determined essentially as previously described33 . Each 1-ml reaction contained 0.2 mg of crude protein, 1.5 mM 3-hydroxybutyryl-CoA substrate and 0.5 mM 5,5-dithiobis(2-nitrobenzoic acid) in Tris-glycerol buffer (25 mM Tris-HCl pH 7.5, containing 5% (v/v) glycerol). The reaction was initiated by adding 3-hydroxybutyryl-CoA and incubated at 30 °C for 10 min. The resulting thiobenzoate anion was measured spectrophotometrically at 412 nm. Protein quantification was performed using the Lowry method.
The acetyl-CoA level was determined using the acetyl-CoA assay kit MAK039 (Sigma-Aldrich, St. Louis, MO, USA). Cells were harvested and frozen in liquid nitrogen. Acetyl-CoA was specifically catabolized by the enzyme assay kit according to the manufacturer’s protocol and the resulting product was quantified by its fluorescent intensity (535-nm excitation and 587-nm emission).

Changes in acetyl co-A concentration were assessed in cell lysate using a colorimetric commercially available kit (Sigma, #MAK039) and performed according to manufacturer’s instructions.