RNAi-Mediated Knockdown of Key Autophagy Genes

For RNAi-mediated knockdown of gene expression, we utilized Dharmacon siGENOME SMARTpool reagents [Non-Targeting Control Pool #2 (D-001206-14-05), BECN1 (M-010552-01), ATG5 (M-004374-04), and ATG7 (M-020112-01); Thermo Scientific, Waltham, MA]. Transfections were conducted according to manufacturer’s instructions with modifications described previously [26 ]. Importantly, double-transfection was used as described by Smith et al., to maximize efficiency and duration of knockdown [31 (link)]. iOvCa147-E2 cells were seeded to 6-well plates at a density of 350,000/well whereas the density for CaOV3, OVCAR8, SKOV3, and HeyA8 cells were 200,000/well. Cells were counted and seeded for further experimentation 96 h following initial transfection.