FIGURE 3.

Knockdown of malonyl-CoA synthetase in HEK293T cells by RNA interference. A, Western analysis of mitochondrial extracts from HEK293T cells treated with vehicle only (lane 1), 20 nm ACP siRNA (lane 2), or 75 nm ACSF3 siRNA (lane 3). Loading was normalized on the basis of citrate synthase activity (14.5 units/lane). B, digitalized Western blot data. β-KS, β-ketoacyl synthase. C, malonyl-CoA synthetase activity in mitochondrial extracts. The 100% value for malonyl-CoA synthetase activity corresponds to 2.9 pmol/min, normalized for 1 unit of citrate synthase activity. D, analysis of ACP-linked thioesters formed from [2-¹⁴C]malonate metabolism by mitochondrial preparations derived from siRNA-treated HEK293T cells. The metabolites detected included malonyl-ACP and its decarboxylation product, acetyl-ACP, and the two major products of fatty acid synthesis, octanoyl-ACP and hexanoyl-ACP. The 100% value for the [¹⁴C]acetyl-ACP plus [¹⁴C]malonyl-ACP (¹⁴C Acet & Mal-ACP) pool size was 6.4 μm; acetyl-ACP accounted for 35 ± 7% of the total pool. The 100% value for [¹⁴C]hexanoyl-ACP was 0.51 μm, and that for [¹⁴C]octanoyl-ACP was 0.62 μm, both expressed in terms of malonyl moieties. The data represent means of at least two experiments, with probability values of Student's t test, when <0.1, shown.