Fig. 4.

LXR activation increases acyl-CoA synthetase activity in BeWo cells, and ACSL3 silencing prevents increased acyl-CoA synthetase activity. BeWo cells were either treated directly or reverse-transfected with ACSL3 siRNA or negative control siRNA and incubated for 48 h in medium supplemented with vehicle (0.2% DMSO), 9-cis RA (1 μM), and/or GW3965 (1 μM). A: In cell lysates harvested from BeWo cells prestimulated with vehicle, 9-cis RA (1 μM), and/or GW3965 (1 μM) for 48 h. Acyl-CoA synthetase activity was analyzed in BeWo cell lysates by incubating the lysates in the presence of 100 μM [¹⁴C]OA for 8 min at 37°C. Generated [¹⁴C]OA-CoA was extracted, and the radioactivity was determined in a scintillation counter. Each experiment was performed in triplicate. The results are the means of three independent experiments performed in triplicate (n = 3) ± SEM. *P < 0.05 and **P < 0.01 relative to control (one tailed t-test). B: Total RNA was extracted and analyzed using qRT-PCR. ACSL3 mRNA expression (normalized to TBP) was compared in cells transfected with ACSL3 siRNA1 (black bar) or negative control siRNA (white bar). The results are the means of two independent experiments performed in duplicates (n = 2) ± SEM. Reduction in expression (in %) relative to negative control siRNA is indicated. C: ACSL3 and β-actin protein expression in cells transfected with control siRNA or ACSL3 siRNA. Each lane contains cells pooled from three wells (n = 2). The experiment was repeated once with similar results. D: Acyl-CoA synthetase activity in cell lysates from cells transfected with ACSL3 siRNA (black bar) or a negative control siRNA (white bar). The results are the means of three independent experiments performed in triplicate (n = 3) ± SEM. Reduction in expression (in %) relative to negative control siRNA is indicated. E: OA uptake in cells transfected with ACSL3 siRNA (black bar) or a negative control siRNA (white bar). A representative experiment of four independent experiments performed in triplicate is shown (n = 3) ± SEM. OA uptake (in %) relative to negative control siRNA is indicated. ACSL, long-chain acyl-CoA synthetase; BeWo, human placental choriocarcinoma; LXR, liver X receptor; OA, oleic acid; qRT-PCR, quantitative reverse transcription-PCR; siRNA, small interfering RNA.