siRNA Transfection of THP-1 Monocytes

We performed small interfering RNA (siRNA) transfection, as previously described by Al-Roub et al.^{30 (link)}. Briefly, we washed THP-1 monocytic cells and resuspended them in nucleofector solution (100 µl; Amaxa Nucleofector Kit V). We transfected the cells separately with siRNA against ACSL1 (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), scramble siRNA (30 nM; OriGene Technologies, Inc., Rockville, MD, USA), and pmaxGFP (0.5 ug; Amaxa Nucleofector Kit V for THP-1cells, Lonza, Cologne, Germany). We performed all transfection experiments with an Amaxa Cell Line Nucleofector Kit V for monocytic cells (Lonza, city, Germany) using an Amaxa Electroporation System (Amaxa Inc., Cologne, Germany)^{17 (link)}. After 36 h, we treated the siRNA transfected cells with TNFα. Next, after 24 h, we harvested the monocytic cells and conditioned media. Lastly, we assessed the gene knockdown level of ACSL1 using real-time PCR.