Igepal ca 630

For the isolation of total cholesterol (TC), 1 × 10⁶/mL LX-2 cells were lysed by 0.1% NP40 lysis buffer and then lipids were extracted with chloroform:isopropanol (7:11) in a micro-homogenizer. The sample was centrifuged at 13,000× g for 10 min and then air-dried at 50 °C to remove residual organic solvents. The pellets were dissolved in the Cholesterol Assay Buffer (Sigma, St. Louis, MO, USA) and vortexed until the mixture was homogenous. For the isolation of triglycerides (TG), 1 × 10⁶/mL LX-2 cells were lysed by IGEPAL CA-630 (Beyotime, Shanghai, China). The sample was slowly heated to 100 °C in a metal bath for 3 min and then cooled to room temperature (RT). Centrifugation took place at 16,000× g for 2 min to collect the supernatant. The amounts of TG, TC, and FC were measured using a cholesterol test kit (Sigma, St. Louis, MO, USA) and triglyceride detection kit (Sigma, St. Louis, MO, USA), respectively, according to the manufacturer’s instructions. Protein concentrations were quantified by the BCA protein quantitative analysis kit (ThermoFisher, Waltham, MA, USA).

Cells were collected and lysed with cell lysis buffer (RIPA) for western blotting (Beyotime, Haimen, China) containing 150 mM NaCl, 1.0% IGEPAL^® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0. The proteins (30 μg per lane) were separated on 12% SDS-polyacrylamide gels and transferred on a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Immunoblotting of the membrane was performed using the following primary antibodies: anti-CDK6 (ab124821), pRb (s795) (ab47474), Rb (ab24) β-actin (ab8227), all the antibodies were purchased from Abcam (Cambridge MA).