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Materials and methods
Cell culture. NCI-H460, human lung cancer cells, and HCT-8 and HCT-15, human colonic carcinoma cells, were purchased from American Type Culture Collection (Manassas, VA) and maintained in RPMI-1640 medium (Hyclone, UT) containing 10% fetal bovine serum and 2 mM glutamine at 37 °C, 5% CO2. For TOFA (Sigma, MO) exposures, CHIR-265 were grown at 60% of confluence, and TOFA was applied at the indicated concentrations for 24 h.
ACCA silencing. Scrambled and ACCA specific siRNAs [14] were chemically synthesized (Ambion, TX) and delivered into NCI-H460, HCT-8 and HCT-15 cells (3.5 × 104–5 in Opti-MEM I medium) as described previously [17].
Palmitic acid rescues. Palmitic acids were provided with a bovine serum albumin (BSA) complex [14]. Briefly, 4 volumes of 4% fatty acid-free BSA (Hyclone, UT) in 0.9% NaCl were mixed with 1 volume of 5 mM palmitate (Sigma, MO) in ethanol and incubated at 37 °C for 1 h to form 1 mM palmitate–BSA complex. Rescues were exerted by adding the palmitate–BSA complex (100 μM) to the TOFA-treated cells at the indicated time.