siRNA and transfection

MDA-MB-231 cells were transfected with 21–25 nt Atg7 siRNA at 100 nM or negative-control (NC) siRNA Guangzhou RiboBio Co., Ltd., Guangzhou, China) using the Atg7 siRNA kit (cat no. RN:R10043.4) following the manufacturer's protocol once cells had been cultured for 24 h, using Lipofectamine^® RNAiMAX Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The target sequence for Atg7 siRNA (cat no. 1294165753) was GGAGTCACAGCTCTTCCTT, and the primer sequences were as follows: Sense, 5′-GGAGUCACAGCUCUUCCUUdTdT-3′ and antisense, 5′-AAGGAAGAGCUGUGACUCCTdTd-3′. Transfection efficiency was determined by detecting Atg7 expression levels with RT-qPCR. Following transfection for 4 h, the medium was removed from all groups and the cells were washed twice with 0.01 mol/l PBS. Next, the cells were re-cultured in DMEM (high-glucose) at 37°C in a humidified atmosphere containing 5% CO₂. Cells were then separated into 3 groups; i) DHA+Atg7(−) group, MDA-MB-231 cells transfected with Atg7-siRNA and treated with DHA (10 µg/ml); ii) DHA group, MDA-MB-231 cells treated with DHA alone (10 µg/ml); iii) DHA+Rapamycin(−) group, MDA-MB-231 cells treated with DHA (10 µg/ml) and rapamycin (100 nmol/l). Each group was repeated three times in triplicate. The time between transfection and subsequent experimentation was 4 h.