Cell culture and cell lines

All cell lines were incubated at 37 °C and were maintained in an atmosphere containing 5% CO₂. A549, H460, H157 and H1355 cells were purchased from the ATCC. Cells were tested for Mycoplasma (Lonza) using manufacturer’s conditions and were deemed negative. Cells were grown in Dulbecco’s modified Eagles medium (DMEM) plus 10% fetal bovine serum (Hyclone). ACC1-KO clones were maintained in 10% FBS + addition of 200 μM palmitate every 3 days. For proliferation assays via cell counts, cells were plated into 24 well plates in triplicate at 2^E4 cells/well and the following day treated with either DMSO vehicle or ND-608 or ND-646. Cell counts were recorded at days 1, 3, 5 and 7-post treatment. For delipidated media, cells were first seeded in media containing regular 10% FBS and the following day were switched into media containing 20% delipidated FBS (Gemini #900-123) upon treatment. Viability assays were performed using either a WST-1 viability assay (Roche #05015944001) or Cyquant (Life Technologies #{"type":"entrez-nucleotide","attrs":{"text":"C35011","term_id":"2371152","term_text":"C35011"}}C35011) in 96 well culture plates under manufacturers conditions. For quantitation, values from treated cells were divided by values from control treated cells and expressed as percent control. Palmitate (Sigma Aldrich #P5585) rescue experiments were performed in delipidated media by co-treating cells, in 24 well plates, with either ND-608 or ND-646 and 200 μM of palmitate conjugated to BSA. For palmitate-BSA preparation, palmitate was solubilized in 100% Etoh @ 50 mM and combined with 4% fatty acid free BSA (Sigma Aldrich #A8806) in saline at a ratio of 1:4 to make a palmitate concentration of 10 mM. Palmitate-BSA was used at a final concentration of 200 μM.