Hyamine hydroxide

The β-oxidation rate of free fatty acid was measured as previously reported,^{63 (link)} with some modifications. HepG2 cells were cultured in DMEM medium on 6-well plate to a density of 1×10⁶ cells per well. For the assay, add to each well 1ml Krebs-Ringer phosphate buffer containing fatty acid-free BSA (20 mg·ml⁻¹), glucose (2.5 mM), oleic acid (0.4 mM, Sigma), and 1-¹⁴C oleic acid (1.0 μCi·ml⁻¹, GE Healthcare). To start the treatment, NEN (1.0 μM) or vehicle were added into each well, which was then covered by a piece of square filter paper soaked with hyamine hydroxide (Perkin Elmer). After incubation for 3 h, 0.5 ml of 4 M H₂SO₄ was added to each well and then the wells were sealed again for additional 30 min before the filter papers were carefully removed. The ¹⁴CO₂ trapped on each filter paper was measured by a scintillation counter (Beckman LS 5000 TD Liquid Scintillation System).

Gemcitabine, dilazep hydrochloride, thymidine, and high-performance liquid chromatography (HPLC) grade acetonitrile and trifluoroacetic acid (TFA) were purchased from Thermo Fisher Scientific (Waltham, MA). The deaminated Gemcitabine metabolite dFdU was purchased from Sigma-Aldrich (St. Louis, MO). Radiolabeled Gemcitabine (cytosine-2-¹⁴C) (55.0 mCi/mmol), subsequently referred to as [¹⁴C]-Gemcitabine, was purchased from Moravek, Inc. (Brea, CA). CytoScint™ scintillation solution was purchased from MP Biomedicals, LLC (Solon, OH). Hyamine hydroxide was purchased from PerkinElmer (Waltham, MA). All other chemicals were obtained from standard commercial sources.