Mitochondrial Fatty Acid Synthesis Knockdown

All cell cultures were maintained at 37°C and 5% CO₂. HeLa cells were plated in 10-cm cell culture dishes (CellTreat) at a density of 1.5 x 10⁵ cells/mL in DMEM + 10% FBS (Mediatech, Gibco). Knockdown of the mtFASII pathway was achieved using Qiagen Flexitube siRNAs specific for the gene for ACP (NDUFAB1) as described in Parl et al., 2013[25 (link)]. Control cells were transfected with Allstars negative control siRNA (Qiagen). Additionally, as a negative control cells were transfected with siRNAs specific for METTL9, a nuclear gene encoding a mitochondrial protein, HeLa cells were transfected with siRNA using HiPerfect Transfection Reagent (Qiagen). In order to maintain the knockdown, cells were re-transfected after 48 h: cells were trypsinized, resuspended in double their original volume of DMEM + 10% FBS, then plated in a 15-cm dish for an additional 48 h. At 96 h, cells were harvested by trypsinization and centrifugation. Knockdown efficiency was measured after 96 h using real time quantitative RT-PCR. Cell viability was unaltered in the siRNA treated cells as determined by cell count and trypan blue uptake.