Ldh activity assay

Fatty acid synthesis was measured in mouse frozen tissues and human HCC cell lines by incorporation of [U-¹⁴C] acetate into lipids as described [35 (link)]. Briefly, HCC cell lines were cultured in 96-well plates at 2.0 × 10³/well and incubated overnight. After adding specific or scramble siRNA, cells were pulse labelled with [U-¹⁴C] acetate (1 μCi/well) for 48 hours. Each condition was run in triplicate. Lipids were Folch extracted and counted for ¹⁴C. Cholesterol and triglyceride levels in HCC cell lines and mouse specimens were assessed using the Cholesterol Quantitation Kit and the Triglyceride Quantification Kit (BioVision Inc., Mountain View, CA), respectively, following the manufacturer's protocol. Lactate dehydrogenase activity was assessed using the LDH activity assay (BioVision Inc.) according to the manufacturer's instructions.

HSF1 activity in mouse frozen liver tissues was determined using the HSF1 ELISA kit (Enzo Life Sciences, Farmingdale, NY), following the manufacturers’ protocol. Fatty acid synthesis was measured by incorporation of [U-¹⁴C] acetate into lipids as described [38 (link)]. Briefly, lipids were Folch extracted and counted for ¹⁴C. Total cholesterol and triglyceride levels in mouse liver specimens were assessed using the Cholesterol Quantitation Kit and the Triglyceride Quantification Kit (BioVision Inc., Mountain View, CA, USA), respectively, following the manufacturer’s recommendation. Lactate dehydrogenase (LDH) activity was assessed using the LDH activity assay (BioVision Inc.) according to the manufacturer’s instructions.

For the ROS assays, the primary hepatocytes were seeded in a 96-well plate (Corning) and treated with various concentrations of APAP (0, 5, 10, 15, 20, and 40 mM) for 24 h. CellROX Deep Red reagent (Life Technologies) was added 30 min before the end point and then washed out with PBS. The ROS intensities were determined by using an ELISA reader (Ex/Em: 644/655 nm, BioTek, Instruments, Inc., Winooski, VT). Cell viability was assessed by the MTT assay (Sigma-Aldrich). Cytotoxicity was determined using the LDH activity assay (BioVision) according to the manufacturer’s instructions.