Triacylglyceride measurement

Triacylglyceride levels were quantified using the Triglyceride Assay Kit (Abcam, #ab65336) according to the manufacturer’s instructions. Briefly, 1 × 10⁷ Caki-1 cells treated with the indicated cell cycle inhibitors were harvested, washed with cold PBS, and subsequently resuspended in 500 μl of 5% NP-40 solution. After two cycles of heating at 95 °C for 5 min followed by cooling to room temperature to solubilize all triglycerides, the samples were diluted in water and mixed with cholesterol esterase/lipase for 20 min. Then, the triglyceride reaction mix was added and incubated for 60 min at room temperature. The intensity of OD570 nm was measured using a Synergy 2 microplate reader (BioTek). For triacylglyceride measurement in tumor tissues, the tissues were homogenized in a 5 % NP-40 solution and subsequently diluted in water for further analysis. The concentration of triacylglyceride was normalized to the cell number or the weight of the tumor tissue.