Benzothonium hydroxide

We performed BCKA oxidation assays by using [¹⁴C]-KIV as previously described [26 ], with minor modifications. Huh7 cells were plated in 3.5 cm dishes at ∼70–80% confluency. The following day, media was replaced with serum-free culture media with indicated treatments overnight. The following day, media was replaced with amino acid free media with DMSO (vehicle), 7ACC2 (2.5 μM), or MSDC-0602K (25 μM), and incubated for 1 h [¹⁴C]-KIV was added (0.065 μCi), then the dish was quickly placed into an airtight CO₂ collection chamber (Fisher Nalgene® style 2118 jar, fitted with a rubber stopper (Kontes 774,250–0014), and a center-well hanging bucket (Kimble 882,320–0000) containing Whatman filter paper soaked with 50 μL benzothonium hydroxide (Sigma)). After 30 min at 37 °C, the reaction was terminated by 100 μL of 0.5 M H₂SO₄, injected using a syringe. [¹⁴C]-CO₂ liberation was allowed to proceed for an additional 30 min on ice, then the center-well collection bucket was cut and put into a liquid scintillation vial. Production of [¹⁴C]-CO₂ was determined via a scintillation counter and normalized to cellular protein, determined using DC protein assay (Bio-Rad).