2.9 De novo lipogenesis assay

Trans-nonachlor mediated alterations in cellular DNL were determined as previously performed with minor modifications (Howell et al., 2009; Howell III et al., 2016). Briefly, McA cells were exposed to either vehicle (DMSO 0.025%) or trans-nonachlor (20 μM) for 24 hours in serum free DMEM containing fatty acid free BSA (0.5%) with or without insulin stimulation (100 nM) for the final 8 hours. Following exposures, cells were then treated with 2 μCi/ml ¹⁴C-acetate for 3 hours. After ¹⁴C acetate incubation, monolayers were washed twice with ice cold PBS, scraped in 200 μl PBS, and total cellular lipids were extracted with chloroform:methanol (2:1; 1 ml) via the Folch method (Folch et al., 1957). Organic phase (600 μl per sample) radioactivity was measured by liquid scintillation counting and expressed as counts per minute/mg cellular protein. Previous studies in our lab have shown that the majority of ¹⁴C-acetate radioactivity in total cellular lipids is incorporated into cellular triglyceride fractions (Howell et al., 2009).