2.7. siRNA Transfection and Brf1-Luc Reporter Assays

For siRNA knockdown, Brf1 siRNAs, AMPKα siRNA, and a control siRNA (mismatch RNA: mmRNA) were purchased from RiboBio. The sequences of primers and siRNAs used were as previously described [17, 18]. Transfections were performed using Lipofectamine 3000 and OPTI-MEM reagent (Life Technologies, Cat No. L3000015 and 11058021) when cells were approximately 40% confluent and transfected according to the manufacturer's instruction [37]. For Brf1-Luc promoter activity, cells were transfected with 0.5 μg of the Brf1-Luc report constructs for 48 h. Cells were starved in FBS-free RPMI 1640 for 4 h and treated with different concentrations of MNNG for another 2 h. Cell pellets were dissolved in Promega reporter lysis buffer. The luciferase activities of these lysates were determined by a luminometer and the Promega Luciferase Kit (Promega, Cat No. E1910). The luciferase activities of the lysates were normalized to their protein amounts as described [31, 32]. The changes in luciferase activity were compared to the luciferase activity in the absence of MNNG. Means ± SE is at least three independent experiments.