Beclin1 sirna

After SKOV3/DDP cells were grown into 6-well plates and reached 50% confluence, cells were transiently transfected with the Beclin1 small interfering (si)RNA or negative control siRNA (100 pmol) using Lipofectamine 2000 ((Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Briefly, 5 µl (100 pmol) Beclin1 siRNA and 5 µl Lipofectamine 2000 were diluted in 50 µl RPMI-1640 medium, respectively, and were incubated for 5 min at room temperature. Then, the diluted Beclin1 siRNA and Lipofectamine 2000 were mixed and incubated for 15 min at room temperature. Next, the complexes were added to cells and incubated for 6 h at 37°C in a 5% CO₂ incubator. Thereafter, the cells were washed with PBS and suspended in the RPMI-1640 medium plus 10% FBS. After 48 h of transfection, cells were treated or untreated with 100 nM TPL for an additional 24 h and collected for western blot analysis. The Beclin1 siRNA (5′-GGATGACAGTGAACAGTTA-3′) and negative control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) were designed and synthesized by Guangzhou Ribobio Co., Ltd.

The A549 and H1299 cells were transfected with 50 nM of IREB2 siRNA, Beclin1 siRNA, and control siRNA (RIBOBIO, Guangzhou, China), respectively, using riboFECTCP transfection kit (RIBOBIO, Guangzhou, China). The target sequence of IREB2 siRNA was “GGCTCATCAAATAAACTTA,” and the target sequence of Beclin1 siRNA was “GGAGTCTCTGACAGACAAA”. We diluted 5 μL of 20 μM siRNAs with 120 μL riboFECTCP buffer, then added 12 μL of riboFECTCP reagent, mixed it gently, and incubated it at room temperature for 5 min. The transfection mixture was then transferred into 6‐well plates combined with 1863 μL of RPMI1640 media without penicillin–streptomycin. After 2 hours, A549 and H1299 cells were incubated with curcumin for another 24 hours and subjected to western blot analysis.