Fig. 1.

siRNA-induced silencing of ABCA1 increases VLDL TG secretion in rat hepatoma cells. A: McA rat hepatoma cells were transfected with either ABCA1 siRNA (0–100 nM) or two control siRNAs for 72 h. Silencing efficiency was assessed by Western blot analysis for ABCA1 expression. All subsequent experiments used 25 nM siRNA to silence ABCA1. B: Control and ABCA1 siRNA transfected McA cells were incubated with radiolabeling medium (10% FBS, 5 μCi/ml ³H-oleate + 0.8 mM oleate) for 24 h. Lipids were extracted from medium and cells, fractionated by TLC, and radiolabeled TG quantified by liquid scintillation spectroscopy. Cellular protein was quantified and radiolabeled TG content was expressed as ³H-TG dpm/μg cell protein (n = 3). C: Conditioned medium (500 µl; pooled, n = 3) from the experiment in B was fractionated by fast protein liquid chromatography and radiolabel in each fraction was determined. VLDL elution position (fractions 30–35) is denoted. D: Control and ABCA1 siRNA transfected McA cells were radiolabeled with [¹⁴C]glycerol (1μCi/ml) and [³H]acetate (5μCi/ml) in the presence of 0.8 mM oleate and cell and medium was harvested at 0, 1, 3, 6, 12, and 24 h. Lipids were extracted from cells and medium and separated into individual lipid classes by TLC, followed by radiolabel quantification in individual lipid classes by liquid scintillation spectroscopy. FC, free cholesterol; TG, triglyceride. ABCA1 siRNA, closed circle and dashed line; control siRNA, open circle and solid line. Results are expressed as mean ± SEM of triplicate wells. * (P < 0.05) and ** (P < 0.01) by Student's t-test. ns, not significant.